Methods for treating a tumor using an antibody that specifically binds grp94

ABSTRACT

Combinations of agents that have a synergistic effect for the treatment of a tumor are disclosed herein. These combinations of agents can be used to treat tumors, wherein the cells of the cancer express a mutated BRAF. Methods are disclosed for treating a subject diagnosed with a tumor that expresses a mutated BRAF. The methods include administering to the subject (1) a therapeutically effective amount of an antibody or antigen binding fragment thereof that specifically binds glucose regulated protein (GRP) 94; and (2) a therapeutically effective amount of a BRAF inhibitor. In some embodiments, the tumor is melanoma. In some embodiments the method includes selecting a subject with primary or secondary resistance to a BRAF inhibitor. In further embodiments, treating the tumor comprises decreasing the metastasis of the tumor. In additional embodiments, the BRAF inhibitor comprises PLX4032 or PLX4720.

PRIORITY CLAIM

This is a continuation of U.S. patent application Ser. No. 13/309,490,filed on Dec. 1, 2011, which claims the benefit of U.S. ProvisionalApplication No. 61/419,208, filed Dec. 2, 2010. Both of the priorapplications are incorporated herein by reference in their entirety.

FIELD

This application relates to the treatment of cancer, specifically to theuse of a combination of an antibody that specifically binds glucoseregulated protein (GRP) 94 and a BRAF inhibitor.

BACKGROUND

Melanoma is a malignant tumor of melanocytes that are predominatelyfound in skin, but can also be found in the bowel and eye. Althoughmelanoma is not the most common form of skin cancer, it causes themajority of skin cancer related deaths. Melanoma incidence and mortalityrates in fair-skinned populations are increasing worldwide.Approximately 160,000 cases of malignant melanoma are diagnosed in theworld each year. Current treatments include surgical removal of thetumor, adjuvant treatment, chemotherapy, immunotherapy and radiationtherapy.

RAF protein kinases are key components of signal transduction pathwaysby which specific extracellular stimuli elicit precise cellularresponses in mammalian cells. Activated cell surface receptors activateras/rap proteins at the inner aspect of the plasma-membrane which inturn recruit and activate Raf proteins. Activated RAF proteinsphosphorylate and activate the intracellular protein kinases MEK1 andMEK2. In turn, activated MEKs catalyze phosphorylation and activation ofp42/p44 mitogen-activated protein kinase (MAPK). Several cytoplasmic andnuclear substrates of activated MAPK are known that directly orindirectly contribute to the cellular response to environmental change.Three distinct genes have been identified in mammals that encode Rafproteins: ARAF, BRAF and CRAF (also known as RAF-1).

Inhibitors of RAF kinases have been suggested for use in disruption oftumor cell growth and in the treatment of cancers, such as histiocyticlymphoma, adenocarcinoma, small cell lung cancer, melanoma andpancreatic and breast carcinoma. Specific inhibitors of BRAF mutants,such as V600E (BRAF^(V600E) mutant) are known and have been used for thetreatment of cancer. However, some subjects are refractory to treatmentwith BRAF inhibitors. Furthermore, some subjects develop secondaryresistance to BRAF inhibitors, such that regression induced by a BRAFinhibitor is only temporary. Thus, a need remains for agents thataugment the effect of BRAF inhibitors, such as a combination of agentsof use for treating cancer and for inhibiting secondary resistance to aBRAF inhibitor.

SUMMARY

Combinations of agents that have a synergistic effect for the treatmentof cancer are disclosed herein. These combinations of agents can be usedto treat cancers, wherein the cells of the cancer express a mutatedBRAF. The compositions include a therapeutically effective amount of aBRAF inhibitor.

Methods are disclosed for treating a subject diagnosed with a tumor thatexpresses a mutated BRAF. The methods include administering to thesubject (1) a therapeutically effective amount of an antibody or antigenbinding fragment thereof that specifically binds glucose regulatedprotein (GRP) 94; and (2) a therapeutically effective amount of a BRAFinhibitor.

In some embodiments, the tumor is melanoma. In further embodiments,treating the tumor comprises decreasing the metastasis of the tumor. Inadditional embodiments, the BRAF inhibitor is vemurafenib (PLX4032). Thedisclosed methods are of use to treat a subject that has primary orsecondary resistance to the BRAF inhibitor. The cells in the tumor canhave a BRAF V600E mutation.

In some embodiments, the methods include administering to the subject atherapeutically effective amount of an antibody or antigen bindingfragment thereof that specifically binds GRP94. For example, theantibody can be a monoclonal antibody, wherein the heavy chain of theantibody comprises the amino acid sequence set forth as amino acids26-33 of SEQ ID NO: 3 (CDR1), amino acids 51-58 of SEQ ID NO: 3 (CDR2),and amino acids 97-103 of SEQ ID NO: 3 (CDR3) and/or wherein the lightchain of the antibody comprises the amino acid sequence set forth asamino acids 27-32 of SEQ ID NO: 4 (CDR1), amino acids 50-52 of SEQ IDNO: 4 (CDR2), and amino acids 89-97 of SEQ ID NO: 4 (CDR3).

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of BRAF-I on GRP94 Expression by M21 Melanoma Cell Line.

Cells (2×10⁵/ml) were incubated in RPMI 1640 medium containing 10% FCSwith 1 μM of BRAF-I for 14 days. Cells were harvested, stained withGRP94-specific fully human antibody (mAb) W9, human IgG (HIg) asnegative control, and analyzed by flow cytometry. Untreated cells wereused as control. Percentage of stained cells and mean fluorescenceintensity (MFI) are indicated.

FIG. 2. GRP94 Expression in BRAF-I Resistant M21 Melanoma Cell Line.

The melanoma cell line M21 acquired resistance to BRAF-I PLX4720following repeated exposures to this inhibitor namely M21R. BRAF-IPLX4720 resistant M21R cells (red) and parental melanoma cells M21(black) were cell surface stained with GRP94-specific mAb W9. Humanimmunoglobulin (HIg) was used as control. Stained cells were analyzedwith a flow cytometer. Percentage of stained cells and mean fluorescenceintensity (MFI) are indicated.

FIG. 3. Synergic Anti-Cell Growth Effect of GRP94-Specific mAb W9Combined with BRAF-I PLX 4720 on Melanoma Cells.

Human melanoma cells (MV3, M21R) were seeded (2.5×10³ cells per well) ina 96-well plate (RPMI 1640 media plus 1% FCS) and treated withGRP94-specific mAb W9, HIg (negative control) in presence of BRAFinhibitor PLX4720 (5 μM) for 1, 2, 3, 4, 5 days at 37° C. in a 5% CO2atmosphere. Cells were then tested by MTT assay. The O.D. values at 540nm indicate the living cells. *p value<0.05; **p value<0.01 (W9 vsW9+BRAF-I).

FIG. 4. Synergic Anti-Cell Growth Effect of GRP94-Specific mAb W9Combined with BRAF-I PLX 4032 on Melanoma Cells.

Human melanoma cells (M21, M21R, Colo38, Colo38R) were seeded (2.5×10³cells per well) in a 96-well plate (RPMI 1640 media plus 1% FCS) andtreated with GRP94-specific mAb W9, HIg (negative control) in presenceof BRAF inhibitor PLX4032 (500 nM) for 1, 3, 5 days at 37° C. in a 5%CO2 atmosphere. Cells were then tested by MTT assay. The O.D. values at540 nm indicate the living cells. *p value<0.05; **p value<0.01 (W9 vsW9+BRAF-I).

FIG. 5. Inhibition by GRP94-Specific Antibody W9 Combined with BRAFInhibitor PLX4720 of M21R Cells Migration.

M21R cells were incubated in RPMI 1640 medium containing 1% FCS with W9Ab, HIg, BRAF Inhibitor PLX4720 (BRAF-I) combined with W9 Ab, BRAF-Icombined with HIg, or BRAF-I in a 24-trans-well plate (2.5×10⁴ per well)for 3 days in a migration assay. Cells incubated in RPMI 1640 mediumcontaining 1% FCS was used as a reference for 100% cell migration. Theresults are expressed as % inhibition of migration, utilizing the valuesobtained in RPMI 1640 medium containing 1% FCS without Ab as areference. *p value<0.05; **p value<0.01.

FIG. 6. Inhibition of Melanoma Cell M21R Signaling Pathways RAS-MEK-ERKand FAK by BRAF-I in Combination with GRP94-Specific Ab W9.

The human melanoma cell M21R was serum starved for 3 days then seeded atthe concentration of 1.0×10⁵ per well in a 6-well plate in RPMI 1640medium without serum and incubated with W9 Ab, the human IgG (HIg), oruntreated in presence of BRAF inhibitor PLX4720 (5 μM) for 72 hours at37° C. Cell lysate were tested in Western blot with anti-RAS, c-RAF,phosphorylated (p)-MEK, p-ERK1/2, ERK1/2, (p)-FAK (Tyr397), FAK, andPKCα mAbs. Calnexin and β-actin was used as the loading control. Thedensity of resultant bands was determined with IMAGEJ™ software,normalized to that of Calnexin and β-actin, shown below the respectivebands. Data are expressed as the percentage of the expression inuntreated control cells.

FIG. 7. Synergic Pro-Apoptosis of GRP94-Specific mAb W9 Combined WithBRAF-I PLX 4032 on Melanoma Cells.

Human melanoma cells (M21 and M21R) were starved for 12 hours and seededat a density of 2×105/ml in a 6-well plate and treated for 6 hours withBRAF-I PLX4032 (500 nM) and Grp94-specific mAb W9 (20 μg/ml) in RPMI1640 medium plus 1.5% FCS. Cells were stained with Annexin V-FITC andPI, and evaluated for apoptosis by flow cytometry according to themanufacturer's protocol (BD PharMingen, San Diego, Calif., USA). Theearly apoptotic cells (annexin V-positive, PI-negative) were determinedusing a flow cytometer.

FIG. 8. Synergic Inhibition by Grp94-Specific mAb W9 Combined withBRAF-I of BRAFV600E Mutant and BRAF-I Resistant Melanoma CICsProliferation In Vitro.

Cells growing in the exponential phase were seeded at a density of2×105/ml. The cells were treated for 3 days with BRAF-I PLX4032 (500 nM)and Grp94-specific mAb W9 (20 μg/ml) in RPMI 1640 medium plus with 1.5%FCS. Cells were stained with ALDEFLUOR® according to the manufacturer'sprotocol (Stem Cell Technologies). Incubation of cells with ALDEFLUOR®in the presence the ALDH1-specific inhibitor diethylaminobenzaldehyde(DEAB), was used as a negative staining control for the assay. Thencells were stained with ABCB5-specific mAb RK1(1 μg/ml) for 30 mM at 4°C., and incubated with APC-conjugated secondary mAb (1:200) (JacksonImmunoresearch).

FIG. 9. Synergic Targeting Multiple Signaling Pathways by GRP94-SpecificmAb W9 Combined with BRAF-I PLX 4032 on Melanoma Cells.

Cells growing in the exponential phase were seeded at a density of2×10⁵/ml. The cells were treated for 3 days with BRAF-I PLX4032 (500 nM)and Grp94-specific mAb W9 (5 μg/ml) in RPMI 1640 medium plus with 2%FCS. Then the cells were collected and lysed in lysis buffer [10 mMTris-HCl [pH 8.2], 1% NP40, 1 mM EDTA, 0.1% BSA, 150 mM NaCl) containing1/50 (vol/vol) of protease inhibitor cocktail (Calbiochem, La Jolla,Calif.)]. Equal amount of proteins (80 μg per well) were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to polyvinylidene fluoride (PVDF) membrane of 0.45 μm poresize (Millipore, Bedford, Mass.). After blocking the membranes with 5%nonfat dry milk plus 2% BSA at room temperature for 2 hrs, membraneswere incubated overnight at 4° C. with anti-RAS, c-RAF, phosphorylated(p)-MEK (Ser217/221), p-ERK (Thr202/Tyr204), p-AKT, PI3 Kp110α, cleavedPARP, SHg, GLI and β-actin mAb. The appropriate peroxidase-conjugatedsecondary mAb (Cell signaling technology) was added and incubation wascontinued at room temperature for an additional 1 hr. After washing themembrane, the bound antibodies were detected using ECL PLUS™ WesternBlotting Detection System (GE Healthcare, Buckinghamshire, UK), andbands were visualized using the FOTO/ANALYST® Investigator EclipseSystem (Fotodyne Incorporate, Hartland, Wis.). The β-actin was used asthe protein loading control.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file[8123-86316-06_Sequence_Listing.txt, May 2, 2014, 21.8 KB], which isincorporated by reference herein.

The nucleic and amino acid sequences listed are shown using standardletter abbreviations for nucleotide bases, and three letter code foramino acids, as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 is the amino acid sequence of GRP94.

SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding GRP94.

SEQ ID NO: 3 is the amino acid sequence of a heavy chain of an antibodythat specifically binds GRP94.

SEQ ID NO: 4 is the amino acid sequence of a light chain of an antibodythat specifically binds GRP94.

SEQ ID NO: 5 is the amino acid sequence of a Pseudomonas exotoxin.

SEQ ID NOs: 6-7 are the amino acid sequence of Pseudomonas exotoxinmotifs.

DETAILED DESCRIPTION

Disclosed herein are methods for treating a tumor in a subject. Themethods include selecting a subject with a BRAF mutation, andadministering to the subject a therapeutically effective amount of 1 anantibody that specifically binds glucose regulated protein (GRP) 94; and2) a BRAF inhibitor. The use of a combination of an antibody thatspecifically binds GRP94 and a BRAF inhibitor for the treatment ofcancer provides an unexpectedly superior result for the treatment of atumor, wherein cells in the tumor comprise a BRAF mutation. In someembodiments, the tumor is a melanoma. In other embodiments, the BRAFmutation is a V600E mutation. In additional embodiments, the subject hasresistance to the BRAF inhibitor. In some specific non-limitingexamples, the BRAF inhibitor is PLX4032 or PLX4720.

Disclosed herein are methods to treat a subject diagnosed with a tumor,such as a tumor that expresses GRP94. Methods are also provided fortreating a melanoma. Melanoma includes spreading melanoma, nodularmelanoma, acral lentiginous melanoma, and lentigo maligna (melanoma).However, the methods disclosed herein can also be used to treat othercancers, such breast cancer, prostate cancer, ovarian cancer, thyroidcancer, colon cancer, stomach cancer, pancreatic cancer, glioma,chordoma, chondrosarcoma, glioma or a squamous cell carcinoma. Squamouscell carcinomas include, but are not limited to head and neck squamouscell carcinoma, and squamous cell cancers of the skin, lung, prostate,esophagus, vagina and cervix.

In some embodiments, the disclosed methods can also be used to preventmetastasis or decrease the number of micrometastases, such asmicrometastases to regional lymph nodes.

Pharmaceutical compositions are also provided that include atherapeutically effective amount of an antibody that specifically bindsGRP94 and a therapeutically effective amount of a BRAF inhibitor. Inspecific, non-liming examples, the BRAF inhibitor is PLX4032 or PLX4720.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand specifically binds an epitope of an antigen, such as GRP94, or afragment thereof. Antibodies are composed of a heavy and a light chain,each of which has a variable region, termed the variable heavy (V_(H))region and the variable light (V_(L)) region. Together, the V_(H) regionand the V_(L) region are responsible for binding the antigen recognizedby the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies), heteroconjugate antibodies (suchas, bispecific antibodies). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3^(rd) Ed., W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined (see, Kabat et al., Sequencesof Proteins of Immunological Interest, U.S. Department of Health andHuman Services, 1991, which is hereby incorporated by reference). TheKabat database is now maintained online. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species, such as humans. The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds GRP94 will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds GRP94.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate.

In another embodiment, a high binding affinity is measured by acompetition radioimmunoassay. In another embodiment, binding affinity ismeasured by ELISA. An antibody that “specifically binds” an antigen,such as GRP94 with a high affinity and does not significantly bind otherunrelated antigens.

BRAF: A member of the Raf kinase family of serine/threonine-specificprotein kinases. This protein plays a role in regulating the MAPkinase/ERKs signaling pathway, which affects cell division,differentiation, and secretion. BRAF transduces cellular regulatorysignals from Ras to MEK in vivo. BRAF is also referred to as v-rafmurine sarcoma viral oncogene homolog B1.

BRAF Mutant: A mutated form of BRAF that has increased basal kinaseactivity relative to the basal kinase activity of wild type BRAF is alsoan activated form of BRAF. More than 30 mutations of the BRAF gene thatare associated with human cancers have been identified. The frequency ofBRAF mutations in melanomas and nevi are 80%. In 90% of the cases, a Glufor Val substitution at position 600 (referred to as V600E) in theactivation segment has been found in human cancers. This mutation isobserved in papillary thyroid cancer, colorectal cancer and melanoma.Other mutations which have been found are R462I, I463S, G464E, G464V,G466A, G466E, G466V, G469A, G469E, N581S, E585K, D594V, F595L, G596R,L597V, T599I, V600D, V600K, V600R, K601E or A728V. Most of thesemutations are clustered to two regions: the glycine-rich P loop of the Nlobe and the activation segment and flanking regions. A mutated form ofBRAF that induces focus formation more efficiently than wild type BRAFis also an activated form of BRAF.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA,IIIB, and IV).

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treating amelanoma or another tumor. In one embodiment, a chemotherapeutic agentis a radioactive compound. One of skill in the art can readily identifya chemotherapeutic agent (see for example, Slapak and Kufe, Principlesof Cancer Therapy, Chapter 86 in Harrison's Principles of InternalMedicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff,Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc; Baltzer,L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed.St. Louis, Mosby-Year Book, 1995; Fischer, D. S., Knobf, M. F.,Durivage, H. J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St.Louis, Mosby-Year Book, 1993). Combination chemotherapy is theadministration of more than one agent to treat cancer. One example isthe administration of an antibody that binds GRP94, used in combinationwith a BRAF inhibitor, such as a chemical compound.

Decrease in survival: As used herein, “decrease in survival” refers to adecrease in the length of time before death of a patient, or an increasein the risk of death for the patient. A decrease in survival also canrefer to a decrease in the average time to death in a group, such as agroup of patients diagnosed with a cancer, such as melanoma.

Diagnosing: Refers to the process of identifying the nature or cause ofa disease or disorder.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an anti-GRP94 antibody, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art (see, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985). Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc,¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I, fluorophores,chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such as GRP94.

Glucose regulated protein (GRP)₉₄ (also known as Endoplasmin): A proteinwhich is the endoplasmic reticulum (ER)-resident member of theheat-shock-protein 90 (Hsp90) family. In vivo, hsp90 and GRP94 interactwith client proteins and function to protect them fromubiquitin-dependent proteasomal degradation. Although the GRP94 proteinis expressed constitutively in all cell types, its expression isup-regulated under various stress conditions including low glucoselevels, low extracellular pH, expression of mutated proteins, and viralinfections. Heat-shock proteins have a cytoprotective function andmodulate apoptosis directly or indirectly.

It has been shown that expression of GRP94 is increased in tumor cells,including hepatocellular carcinoma, colorectal carcinoma and lung cancercells, and that GRP94 has an anti-apoptotic effect on some tumor cells.Moreover, increased levels of GRP94 were observed when a chronichepatitis B virus (HBV) infection progressed to cirrhosis andhepatocellular carcinoma (HCC) Inhibitors of Hsp90 and GRP94 (such asgeldanamycin (GA) and its less toxic derivative 17-AAG) have beeninvestigated for efficacy in cancer treatment.

GRP94 (endoplasmin) may be encoded by the following genes, but notlimited thereto: GENBANK® Accession Nos. NM_(—)003299, BC066656 (Homosapiens); NM_(—)011631 (Mus musculus); NM_(—)001045763: (Xenopus(Silurana) tropicalis); NM_(—)214103 (Sus scrofa) NM_(—)98210 (Daniorerio); NM_(—)001012197 (Rattus norvegicus); NM_(—)001134101: Pongoabelii; NM_(—)001003327 (Canis lupus familiaris) heat shock protein 90kDa beta (GRP94); NM_(—)204289 (Gallus gallus).

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, pap, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Inhibitor: As used herein, an “inhibitor” refers to any compound that iscapable of reducing or altering the expression or activity of a targetmolecule. In some embodiments, the inhibitor is an inhibitor of BRAF.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Melanoma: A form of cancer that originates in melanocytes (cells thatmake the pigment melanin). Melanocytes are found primarily in the skin,but are also present in the bowel and eye. As used herein, “melanoma”refers to any stage of melanoma, or any subtype of melanoma, such assuperficial spreading melanoma, nodular melanoma, acral lentiginousmelanoma, lentigo maligna, melanoma-in-situ, mucosal melanoma and uvealmelanoma. Melanoma in the skin includes superficial spreading melanoma,nodular melanoma, acral lentiginous melanoma, and lentigo maligna(melanoma). Any of the above types may produce melanin or can beamelanotic. Similarly, any subtype may show desmoplasia (dense fibrousreaction with neurotropism) which is a marker of aggressive behavior anda tendency to local recurrence. Other melanomas include clear cellsarcoma, mucosal melanoma and uveal melanoma.

Features that affect prognosis are tumor thickness in millimeters(Breslow's depth), depth related to skin structures (Clark level), typeof melanoma, presence of ulceration, presence of lymphatic/perineuralinvasion, presence of tumor infiltrating lymphocytes (if present,prognosis is better), location of lesion, presence of satellite lesions,and presence of regional or distant metastasis. When melanomas havespread to the lymph nodes, one of the most important factors is thenumber of nodes with malignancy. The extent of malignancy within a nodeis also important; micrometastases in which malignancy is onlymicroscopic have a more favorable prognosis than macrometastases. Whenthere is distant metastasis, the five year survival rate is less than 10percent; the median survival is 6 to 12 months. Metastases to skin andlungs have a better prognosis. Metastases to brain, bone and liver areassociated with a worse prognosis.

Melanoma can be staged as follows:

-   -   Stage 0: Melanoma in Situ (Clark Level I), 100% Survival    -   Stage I/II: Invasive Melanoma, 85-95% Survival        -   T1a: Less than 1.00 mm primary, w/o Ulceration, Clark Level            II-III        -   T1b: Less than 1.00 mm primary, w/Ulceration or Clark Level            IV-V        -   T2a: 1.00-2.00 mm primary, w/o Ulceration    -   Stage II: High Risk Melanoma, 40-85% Survival        -   T2b: 1.00-2.00 mm primary, w/Ulceration        -   T3a: 2.00-4.00 mm primary, w/o Ulceration        -   T3b: 2.00-4.00 mm primary, w/Ulceration        -   T4a: 4.00 mm or greater primary w/o Ulceration        -   T4b: 4.00 mm or greater primary w/Ulceration    -   Stage III: Regional Metastasis, 25-60% Survival        -   N1: Single Positive Lymph Node        -   N2: 2-3 Positive Lymph Nodes OR Regional Skin/In-Transit            Metastasis        -   N3: 4 Positive Lymph Nodes OR Lymph Node and Regional            Skin/In Transit Metastases    -   Stage IV: Distant Metastasis, 9-15% Survival        -   M1a: Distant Skin Metastasis, Normal lactate dehydrogenase            (LDH)        -   M1b: Lung Metastasis, Normal LDH        -   M1c: Other Distant Metastasis OR Any Distant Metastasis with            Elevated LDH

Metastasis: Refers to the spread of cancer cells from the original tumorto other sites in the body.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized and fullyhuman monoclonal antibodies. As used herein a monoclonal antibodyincludes antibody fragments, such as, but not limited to scFv, Fv, dsRv,or Fab.

Mutation: Any change of the DNA sequence within a gene or chromosome. Insome instances, a mutation will alter a characteristic or trait(phenotype), but this is not always the case. Types of mutations includebase substitution point mutations (e.g., transitions or transversions),deletions and insertions. Missense mutations are those that introduce adifferent amino acid into the sequence of the encoded protein; nonsensemutations are those that introduce a new stop codon. In the case ofinsertions or deletions, mutations can be in-frame (not changing theframe of the overall sequence) or frame shift mutations, which mayresult in the misreading of a large number of codons (and often leads toabnormal termination of the encoded product due to the presence of astop codon in the alternative frame).

This term specifically encompasses variations that arise through somaticmutation, for instance those that are found only in disease cells (suchas cancer cells), but not constitutionally, in a given individual.Examples of such somatically-acquired variations include the pointmutations that frequently result in altered function of various genesthat are involved in development of cancers. This term also encompassesDNA alterations that are present constitutionally, that alter thefunction of the encoded protein in a readily demonstrable manner, andthat can be inherited by the children of an affected individual. In thisrespect, the term overlaps with “polymorphism,” as discussed below, butgenerally refers to the subset of constitutional alterations that havearisen within the past few generations in a kindred and that are notwidely disseminated in a population group. In some embodiments, amutation in BRAF refers to a nucleotide substitution in the BRAF gene orcDNA, or an amino acid substitution in the BRAF protein.

Neoplasia, malignancy, cancer or tumor: The result of abnormal anduncontrolled growth of cells. A neoplasm is an abnormal growth of tissueor cells that results from excessive cell division. Neoplastic growthcan produce a tumor.

malignancy, cancer and tumor are often used interchangeably. The amountof a tumor in an individual is the “tumor burden” which can be measuredas the number, volume, or weight of the tumor. A tumor that does notmetastasize is referred to as “benign.” A tumor that invades thesurrounding tissue and/or can metastasize is referred to as “malignant.”A “non-cancerous tissue” is a tissue from the same organ wherein themalignant neoplasm formed, but does not have the characteristicpathology of the neoplasm. Generally, noncancerous tissue appearshistologically normal. A “normal tissue” is tissue from an organ,wherein the organ is not affected by cancer or another disease ordisorder of that organ. A “cancer-free” subject has not been diagnosedwith a cancer of that organ and does not have detectable cancer.

Examples of hematological tumors include leukemias, including acuteleukemias (such as 11q23-positive acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute myelogenous leukemia andmyeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,medullary thyroid carcinoma, papillary thyroid carcinoma,pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma,medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma and retinoblastoma).

In several examples, a tumor is melanoma, breast cancer, prostatecancer, glioma or a squamous cell carcinoma, such as head and neckcancer.

Nevi: Melanocytic lesions that can be considered regional melanocytichyperplasias. The term “nevi” includes all types of nevi, such ascongenital nevi, acquired nevi, intradermal nevi, compound nevi,dysplastic nevi, atypical nevi, and junctional nevi.

Nevus: The term “nevus” encompasses one or more nevi, including one ormore in vivo nevi cells and one or more in vitro nevi cells. A “nevus”also encompasses one or more melanocytic lesions that can be consideredregional melanocytic hyperplasias. The term “nevus” as used hereinincludes all types of nevi, such as congenital nevi, acquired nevi,intradermal nevi, compound nevi, dysplastic nevi, atypical nevi, andjunctional nevi.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, bythe homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see for example, Current Protocols in Molecular Biology(Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi nlm nih.gov/). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Patient: As used herein, the term “patient” includes human and non-humananimals. The preferred patient for treatment is a human.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds, molecules or agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Polymorphism: Variant in a sequence of a gene, or any genomic sequence,usually carried from one generation to another in a population.Polymorphisms can be those variations (nucleotide sequence differences)that, while having a different nucleotide sequence, produce functionallyequivalent gene products, such as those variations generally foundbetween individuals, different ethnic groups, and geographic locations.The term polymorphism also encompasses variations that produce geneproducts with altered function, i.e., variants in the gene sequence thatlead to gene products that are not functionally equivalent. This termalso encompasses variations that produce no gene product, an inactivegene product, a truncated gene product, or increased or increasedactivity gene product.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule or protein that islinked to the variation (e.g., an alteration of a secondary structuresuch as a stem-loop, or an alteration of the binding affinity of thenucleic acid for associated molecules, such as polymerases, RNAses, achange in the availability of a site for cleavage by a restrictionendonuclease, either the formation of a new site, or lose of a site, andso forth).

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

Conservative amino acid substitutions are those substitutions that, whenmade, least interfere with the properties of the original protein, thatis, the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. Examplesof conservative substitutions are shown in the following table:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Conservative substitutions generally maintain (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

The substitutions which in general are expected to produce the greatestchanges in protein properties will be non-conservative, for instancechanges in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted for (or by) a hydrophobic residue, for example,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, for example, lysyl, arginyl, orhistadyl, is substituted for (or by) an electronegative residue, forexample, glutamyl or aspartyl; or (d) a residue having a bulky sidechain, for example, phenylalanine, is substituted for (or by) one nothaving a side chain, for example, glycine.

Preventing, treating or ameliorating a disease: “Preventing” a disease(such as metastatic melanoma) refers to inhibiting the full developmentof a disease. “Treating” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. “Ameliorating” refers to the reduction inthe number or severity of signs or symptoms of a disease.

Prognosis: The likelihood of the clinical outcome for a subjectafflicted with a specific disease or disorder. With regard to cancer,the prognosis is a representation of the likelihood (probability) thatthe subject will survive (such as for one, two, three, four or fiveyears) and/or the likelihood (probability) that the tumor willmetastasize. A “poor prognosis” indicates a greater than 50% chance thatthe subject will not survive to a specified time point (such as one,two, three, four or five years), and/or a greater than 50% chance thatthe tumor will metastasize. In several examples, a poor prognosisindicates that there is a greater than 60%, 70%, 80%, or 90% chance thatthe subject will not survive and/or a greater than 60%, 70%, 80% or 90%chance that the tumor will metastasize. Conversely, a “good prognosis”indicates a greater than 50% chance that the subject will survive to aspecified time point (such as one, two, three, four or five years),and/or a greater than 50% chance that the tumor will not metastasize. Inseveral examples, a good prognosis indicates that there is a greaterthan 60%, 70%, 80%, or 90% chance that the subject will survive and/or agreater than 60%, 70%, 80% or 90% chance that the tumor will notmetastasize.

Proliferation: One or more cellular events that result in cell growth.Proliferation includes any of a number of growth activities includingincrease in the number of cells, increase in the rate of cell division,increase in the number of cell divisions, increase in the size of acell, change in cellular differentiation, transformation to a malignantstate, metastatic transformation, change in cell cycle phase to a moremitotically active cell cycle phase (e.g., S phase), or a combination oftwo or more of those activities. Cell growth (either in vitro or invivo) can be a hyper-proliferative condition, such as is characteristicof certain disorders or diseases, for instance neoplasia or tumorformation.

Inhibiting proliferation includes any of a number of anti-growthactivities that reduce or even eliminate the ability of a cell toproliferate Inhibiting proliferation includes, for instance, decreasingcell number, decreasing colony forming ability, decreasing the rate ofcell division, decreasing the number of cell divisions, stopping celldivision, inducing apoptosis, inducing senescence, inducing quiescence,changing cell cycle phase to a less mitotically active cell cycle phase,decreasing cellular de-differentiation, preventing transformation to amalignancy, decreasing malignant potential, decreasing metastaticability or potential or a combination of two or more of thoseactivities.

Resistance: The lack of response of a disease, such as a cancer, to atherapeutic agent. In some embodiment, the agent is a BRAF inhibitor.Primary resistance is the lack of a response to a therapeutic agent uponinitial treatment with the agent. Secondary resistance is the lack of aresponse to a therapeutic agent, wherein the cancer in the subject isinitially susceptible to treatment with the agent. Resistance of acancer to an agent can be measured by an increase in tumor burden, anincrease in the number of metastases, or an increase in the amount of atumor marker present in the subject.

Sample: A biological specimen containing genomic DNA, RNA, protein, orcombinations thereof, obtained from a subject. Examples include, but arenot limited to, peripheral blood, urine, saliva, tissue biopsy (such asskin tissue), surgical specimen, and autopsy material. In one example, asample includes a biopsy of a melanoma tumor or a sample of normaltissue, such as skin tissue (from a subject not afflicted with a knowndisease or disorder, such as a cancer-free subject).

Somatic mutation: An acquired mutation that occurs in a somatic cell (asopposed to a germ cell).

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals. In some embodiments, thesubject is a human subject.

Therapeutic agent: A chemical compound, small molecule, or othercomposition, such as an antisense compound, antibody, peptide or nucleicacid molecule capable of inducing a desired therapeutic or prophylacticeffect when properly administered to a subject. For example, therapeuticagents for melanoma include agents that prevent or inhibit developmentor metastasis of melanoma. In some embodiments, the therapeutic agent isan inhibitor of BRAF.

Therapy: The mode of treatment or care of a patient. In some cases,therapy refers to administration of a therapeutic agent. In someembodiments herein, therapy includes administration of a BRAF inhibitor.In other examples, therapy includes surgery, such as surgical resectionof a melanoma tumor, chemotherapy, radiation therapy, or any combinationthereof.

Therapeutically effective amount: A quantity of an agent sufficient toachieve a desired effect in a subject or a cell being treated. Forinstance, this can be the amount necessary to inhibit or to measurablyreduce B-Raf activity in a nevi or to inhibit melanoma proliferation. Atherapeutically effective amount of an agent may be administered in asingle dose, or in several doses, for example daily or more often,during a course of treatment. However, the effective amount will bedependent on the particular agent applied, the subject being treated,the severity and type of the affliction, and the manner ofadministration.

Treating: Includes inhibiting or preventing the partial or fulldevelopment or progression of a disease or medical condition or abnormalbiological state in a subject, for example in a person who is known tohave a predisposition to or to be at risk for the disease or medicalcondition or abnormal biological state, or a cell or a lesion, forinstance a nevus or a melanoma. Furthermore, “treating” refers to atherapeutic intervention that ameliorates at least one sign or symptomof a disease or pathological condition, or interferes with apathophysiological process, after the disease or pathological conditionhas begun to develop. The therapeutic intervention can be prophylacticinhibition of a disease or medical condition or biological state, andtherapeutic interventions to alter the natural course of an untreateddisease process or medical condition or a biological state, such as atumor growth. The therapeutic intervention can be surgical, includingbut not limited to cryosurgery or ablation, such as laser ablation, andadministration of agents, systemically, regionally, or topically orlocally.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Antibodies that Specifically Bind GRP94, Antigen Binding Fragments andImmunotoxins

Antibodies have been produced that specifically bind GRP94 (also knownas endoplasmin) including monoclonal antibodies, such as fully humanmonoclonal antibodies. These antibodies and/or antigen binding fragmentsthereof can be used in the methods disclosed herein. These antibodiescan be conjugated to labels or effector molecules. The antibodies usedin the disclosed methods can be fully human monoclonal antibodies andfunctional fragments thereof that specifically bind GRP 94 (endoplasmin)

Exemplary GRP94 antibodies of use in the methods and compositionsdisclosed herein include those listed in the following table:

Name Source Clonality/Source HSP90B1 antibody (Cat. # 60012-1-Ig)Proteintech Group monoclonal, mouse HSP90B1 antibody (Cat. # 14700-1-AP)Proteintech Group polyclonal, rabbit HSP90B1 antibody (Cat. #10979-1-AP) Proteintech Group polyclonal, rabbit Human TRA-1-60 MAb(clone TRA- R & D Systems monoclonal, mouse 1-60) Human TRA-1-85 MAb(clone TRA- R & D Systems monoclonal, mouse 1-85) Human TRA-1-85phycoerythrin R & D Systems monoclonal, mouse MAb (clone TRA-1-85) GRP94polyclonal antibody Enzo Life Sciences polyclonal, rabbit GRP94monoclonal antibody (9G10), Enzo Life Sciences monoclonal, mouse PEGRP94 monoclonal antibody (9G10), Enzo Life Sciences monoclonal, ratDyLight 488 Anti-human TRA1 antibody (clone ATGen monoclonal, mouse 2H3)HSP90B1 antibody Epitomics polyclonal, rabbit HSP90B antibody Epitomicspolyclonal, rabbit GRP94 antibody Epitomics monoclonal, rabbit HSP90B1antibody OriGene polyclonal, goat Anti-GRP94/TRA1 antibody EverestBiotech polyclonal, goat Anti-HSP90B1 antibody AbDSerotec polyclonal,rabbit Anti-human GRP94 antibody AbDSerotec polyclonal, rabbitAnti-human TRA1 antibody MyBioSource polyclonal, goat Anti-human TRA1antibody (clone MyBioSource monoclonal, mouse 2H3) Anti-GRP94 antibodyMyBioSource monoclonal, rat HSP90B1 antibody GeneTex polyclonal, rabbitGRP94 antibody Abcam monoclonal, mouse Alexa Fluor ™ 488 anti-human TRA-BioLegend monoclonal, mouse 1-60-R PE anti-human TRA-1-60-R BioLegendmonoclonal, mouse Heat Shock Protein 94 antibody Thermo Fisherpolyclonal, rabbit Scientific Glucose-Regulated Protein 94 Thermo Fisherpolyclonal, rabbit Scientific Anti-human Heat Shock Protein ProSpecmonoclonal, mouse 90 kDa Beta (GRP94) Member 1 antibody Anti-GRP94StressMarq monoclonal, rat Biosciences GRP94 antibody Abbiotecpolyclonal, rabbit HSP90B1 antibody (N-term) Abgent polyclonal, rabbitGRP94 antibody US Biological polyclonal, rabbit TRA-1-60 antibody USBiological monoclonal, mouse GRP94 antibody US Biological polyclonal,goat GRP94 antibody Novus Biologicals polyclonal, rabbit TRA-1-60antibody (MG38) Novus Biologicals monoclonal, mouse TRA-1-60(S) antibodyCell Signaling monoclonal, mouse Technology TRA-1-85 mAb Cell Signalingmonoclonal, mouse Technology HSP90B1 anti-mouse polyclonal LifeSpanBiosciences polyclonal, rabbit antibody HSP90B1 anti-human polyclonalLifeSpan Biosciences polyclonal, goat antibody HSP90B1 anti-chickenmonoclonal LifeSpan Biosciences monoclonal, rat antibody (9G10) GRP 94(H-212) Santa Cruz polyclonal, rabbit Biotechnology GRP 94 (C-19) SantaCruz polyclonal, goat Biotechnology GRP 94 (4E89) Santa Cruz monoclonal,rabbit Biotechnology Glucose Regulated Protein 94 Biotrend polyclonal,rat antibody Anti-TRA-1-60, clone TRA-1-60 Millipore monoclonal, mouse

Humanized forms and antigen binding fragments of these antibodies arealso of use in the presently disclosed methods.

In one example, human GRP94 (also known as endoplasmin) has an aminoacid sequence set forth as:

SEQ ID NO: 1 MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHINESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTA EKDEL,See also GENBANK ® Accession No. NM 003299 incorporated hereinby reference.

In another example, the GRP94 is encoded by the nucleic acid sequenceset forth as:

SEQ ID NO: 2gtgggcggac cgcgcggctg gaggtgtgag gatccgaacc caggggtggg gggtggaggcggctcctgcg atcgaagggg acttgagact caccggccgc acgccatgag ggccctgtgggtgctgggcc tctgctgcgt cctgctgacc ttcgggtcgg tcagagctga cgatgaagttgatgtggatg gtacagtaga agaggatctg ggtaaaagta gagaaggatc aaggacggatgatgaagtag tacagagaga ggaagaagct attcagttgg atggattaaa tgcatcacaaataagagaac ttagagagaa gtcggaaaag tttgccttcc aagccgaagt taacagaatgatgaaactta tcatcaattc attgtataaa aataaagaga ttttcctgag agaactgatttcaaatgctt ctgatgcttt agataagata aggctaatat cactgactga tgaaaatgctctttctggaa atgaggaact aacagtcaaa attaagtgtg ataaggagaa gaacctgctgcatgtcacag acaccggtgt aggaatgacc agagaagagt tggttaaaaa ccttggtaccatagccaaat ctgggacaag cgagttttta aacaaaatga ctgaagcaca ggaagatggccagtcaactt ctgaattgat tggccagttt ggtgtcggtt tctattccgc cttccttgtagcagataagg ttattgtcac ttcaaaacac aacaacgata cccagcacat ctgggagtctgactccaatg aattttctgt aattgctgac ccaagaggaa acactctagg acggggaacgacaattaccc ttgtcttaaa agaagaagca tctgattacc ttgaattgga tacaattaaaaatctcgtca aaaaatattc acagttcata aactttccta tttatgtatg gagcagcaagactgaaactg ttgaggagcc catggaggaa gaagaagcag ccaaagaaga gaaagaagaatctgatgatg aagctgcagt agaggaagaa gaagaagaaa agaaaccaaa gactaaaaaagttgaaaaaa ctgtctggga ctgggaactt atgaatgata tcaaaccaat atggcagagaccatcaaaag aagtagaaga agatgaatac aaagctttct acaaatcatt ttcaaaggaaagtgatgacc ccatggctta tattcacttt actgctgaag gggaagttac cttcaaatcaattttatttg tacccacatc tgctccacgt ggtctgtttg acgaatatgg atctaaaaagagcgattaca ttaagctcta tgtgcgccgt gtattcatca cagacgactt ccatgatatgatgcctaaat acctcaattt tgtcaagggt gtggtggact cagatgatct ccccttgaatgtttcccgcg agactcttca gcaacataaa ctgcttaagg tgattaggaa gaagcttgttcgtaaaacgc tggacatgat caagaagatt gctgatgata aatacaatga tactttttggaaagaatttg gtaccaacat caagcttggt gtgattgaag accactcgaa tcgaacacgtcttgctaaac ttcttaggtt ccagtcttct catcatccaa ctgacattac tagcctagaccagtatgtgg aaagaatgaa ggaaaaacaa gacaaaatct acttcatggc tgggtccagcagaaaagagg ctgaatcttc tccatttgtt gagcgacttc tgaaaaaggg ctatgaagttatttacctca cagaacctgt ggatgaatac tgtattcagg cccttcccga atttgatgggaagaggttcc agaatgttgc caaggaagga gtgaagttcg atgaaagtga gaaaactaaggagagtcgtg aagcagttga gaaagaattt gagcctctgc tgaattggat gaaagataaagcccttaagg acaagattga aaaggctgtg gtgtctcagc gcctgacaga atctccgtgtgctttggtgg ccagccagta cggatggtct ggcaacatgg agagaatcat gaaagcacaagcgtaccaaa cgggcaagga catctctaca aattactatg cgagtcagaa gaaaacatttgaaattaatc ccagacaccc gctgatcaga gacatgcttc gacgaattaa ggaagatgaagatgataaaa cagttttgga tcttgctgtg gttttgtttg aaacagcaac gcttcggtcagggtatcttt taccagacac taaagcatat ggagatagaa tagaaagaat gcttcgcctcagtttgaaca ttgaccctga tgcaaaggtg gaagaagagc ccgaagaaga acctgaagagacagcagaag acacaacaga agacacagag caagacgaag atgaagaaat ggatgtgggaacagatgaag aagaagaaac agcaaaggaa tctacagctg aaaaagatga attgtaaattatactctcac catttggatc ctgtgtggag agggaatgtg aaatttacat catttctttttgggagagac ttgttttgga tgccccctaa tccccttctc ccctgcactg taaaatgtgggattatgggt cacaggaaaa agtgggtttt ttagttgaat tttttttaac attcctcatgaatgtaaatt tgtactattt aactgactat tcttgatgta aaatcttgtc atgtgtataaaaataaaaaa gatcccaaat, see also GENBANK ®Accession No. NM 003299, incorporated herein by reference.

Once of skill in the art can readily use a nucleic acid sequence toproduce a polypeptide, such as GRP94 using standard method in molecularbiology (see, for example, Molecular Cloning: A Laboratory Manual, 2nded., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989).

Described herein are methods for using isolated human monoclonalantibodies and fragments thereof that specifically bind human GRP94(endoplasmin) for the treatment of cancer, such as, but not limited to,melanoma. In some embodiments, the human monoclonal antibody functionalfragment is a scFv. Also described are compositions including amonoclonal antibody or antigen fragment thereof, a BRAF inhibitor, and apharmaceutically acceptable carrier. Nucleic acids encoding theseantibodies, expression vectors comprising these nucleic acids, andisolated host cells that express the nucleic acids are also provided.Thus, in other embodiments, compositions are provided that include anucleic acid encoding a monoclonal antibody or antigen fragment thereof,a BRAF inhibitor, and a pharmaceutically acceptable carrier

In some embodiments, the human monoclonal antibody or functionalfragment thereof comprises at least a portion of the variable chain ofthe heavy chain amino acid sequence set forth as SEQ ID NO: 3 andspecifically binds GRP94. For example, the human monoclonal antibody caninclude the SDRs (specificity determining residues), the CDRs, or thevariable region of the amino acid sequence set forth as SEQ ID NO: 3. Inthe amino acid sequence shown below, the constant region is in bold, andthe CDRs are underlined:

(SEQ ID NO: 3)

S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G A LT S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V NH K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F LF P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D GV E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E YK C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M TK N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V LD S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y TQ K S L S L S P G K

In some embodiments, the monoclonal antibody or functional fragmentthereof comprises at least a portion of the heavy chain amino acidsequence set forth as SEQ ID NO: 3 and specifically binds GRP94. Themonoclonal antibody can be a human monoclonal antibody. In someexamples, at least one of the CDRs of the light chain of the antibodycomprises one or more of the amino acid sequences set forth as aminoacids 26-33 of SEQ ID NO: 3 (CDR1), amino acids 51-58 of SEQ ID NO: 3(CDR2), and amino acids 97-103 of SEQ ID NO: 3 (CDR3). In additionalexamples, the heavy chain of the antibody comprises the amino acidsequence set forth as amino acids 26-33 of SEQ ID NO: 3 (CDR1), aminoacids 51-58 of SEQ ID NO: 3 (CDR2), and amino acids 97-103 of SEQ ID NO:3 (CDR3). In some examples, the variable region of the heavy chain ofthe antibody can include, or consist of, amino acids 1-113 of SEQ ID NO:3. The heavy chain of the antibody can include, or consist of, SEQ IDNO: 3.

In some embodiments, the monoclonal antibody or functional fragmentthereof comprises at least a portion of the variable region of the lightchain amino acid sequence set forth as SEQ ID NO: 4 and specificallybinds GRP94. The monoclonal antibody can be a human monoclonal antibody.In the amino acid sequence shown below, the constant region is in bold,and the CDRs are underlined:

(SEQ ID NO: 4)

S G T A S V V C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T EQ D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S PV T K S F N R G E CIn some examples, at least one of the CDRs of the light chain of theantibody comprises one or more of the amino acid sequences set forth asamino acids 27-32 of SEQ ID NO: 8 (CDR1), amino acids 50-52 of SEQ IDNO: 4 (CDR2), and amino acids 89-97 of SEQ ID NO: 4 (CDR3). Inadditional examples, the light chain of the antibody comprises aminoacids amino acids 27-32 of SEQ ID NO: 4 (CDR1), amino acids 50-52 of SEQID NO: 4 (CDR2), and amino acids 89-97 of SEQ ID NO: 4 (CDR3). Thevariable region of the light chain of the antibody can include, orconsist of, amino acids 1-107 of SEQ ID NO: 4. The light chain of theantibody can include, or consist of, SEQ ID NO: 4.

Fully human monoclonal antibodies include human framework regions. Thehuman framework regions can include the framework regions disclosed inone or both of SEQ ID NO: 3 or SEQ ID NO: 4 (these sequences include CDRsequences as well as framework sequences). However, the frameworkregions can be from another source. Additional examples of frameworksequences that can be used include the amino acid framework sequences ofthe heavy and light chains disclosed in PCT Publication No. WO2006/074071 (see, for example, SEQ ID NOs: 1-16), which is hereinincorporated by reference.

Chimeric antibodies include CDRs from one species, and framework regions(and/or a constant domain), from another species. In some embodiments,the monoclonal antibody, or antigen binding fragment, is chimeric. Inone specific non-limiting example, the monoclonal antibody includes theCDRs from a murine antibody, and a human framework region. In anotherspecific non-liming example, the monoclonal antibody includes the CDRsfrom a rabbit antibody, and a human framework region.

The monoclonal antibody of use in the disclosed methods can be of anyisotype. The monoclonal antibody can be, for example, an IgA, IgM or anIgG antibody, such as IgG₁ or an IgG₂. The class of an antibody thatspecifically binds GRP94 can be switched with another. In one aspect, anucleic acid molecule encoding V_(L) or V_(H) is isolated using methodswell-known in the art, such that it does not include any nucleic acidsequences encoding the constant region of the light or heavy chain,respectively. The nucleic acid molecule encoding V_(L) or V_(H) is thenoperatively linked to a nucleic acid sequence encoding a C_(L) or C_(H)from a different class of immunoglobulin molecule. This can be achievedusing a vector or nucleic acid molecule that comprises a C_(L) or C_(H)chain, as known in the art. For example, an antibody that specificallybinds GRP94 that was originally IgM may be class switched to an IgG.Class switching can be used to convert one IgG subclass to another, suchas from IgG₁ to IgG₂.

The method disclosed herein can utilize immunoconjugates comprising themonoclonal antibodies or functional fragment thereof that specificallybinds human GRP94, such as human monoclonal antibodies. Theimmunoconjugates can comprise any therapeutic agent, toxin or othermoiety. In one example, the toxin is PE or a variant or fragmentthereof.

Antibody fragments that specifically bind GRP94 are encompassed by thepresent disclosure, such as Fab, F(ab)₂, and Fv which include a heavychain and light chain variable region and are capable of binding theepitopic determinant on GRP94. These antibody fragments retain theability to specifically bind with the antigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of M912.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV2), are also contemplated.

The antibody can also be included in a bi-specific antibody. In furtherembodiments, an engineered antibody domain, including the heavy chainCDRs or the light chain CDRs can be utilized, provided the engineeredantibody domain specifically binds GRP94.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art.

In some embodiments, the disclosed methods utilize immunoconjugates.Immunoconjugates include, but are not limited to, molecules in whichthere is a covalent linkage of a diagnostic or therapeutic agent with anantibody. A therapeutic agent is an agent with a particular biologicalactivity directed against a particular target molecule or a cell bearinga target molecule. Therapeutic agents include various drugs such asvinblastine, daunomycin and the like, and effector molecules such ascytotoxins such as native or modified Pseudomonas exotoxin or Diphtheriatoxin, encapsulating agents (e.g., liposomes), which themselves containpharmacological compositions, target moieties and ligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell and the biological effect desired. Thus, forexample, the therapeutic agent may be an effector molecule that iscytotoxin which is used to bring about the death of a particular targetcell. Conversely, where it is merely desired to invoke a non-lethalbiological response, a therapeutic agent can be conjugated to anon-lethal pharmacological agent or a liposome containing a non-lethalpharmacological agent.

Toxins can be employed with antibodies that bind GRP94 polypeptide andantigen biding antibody fragments, such as a svFv or a dsFv, to yieldchimeric molecules, which are of use as immunotoxins. Exemplary toxinsinclude Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin andsubunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin,as well as botulinum toxins A through F. These toxins are well known inthe art and many are readily available from commercial sources (forexample, Sigma Chemical Company, St. Louis, Mo.).

Diphtheria toxin is isolated from Corynebacterium diphtheriae.Typically, diphtheria toxin for use in immunotoxins is mutated to reduceor to eliminate non-specific toxicity. A mutant known as CRM107, whichhas full enzymatic activity but markedly reduced non-specific toxicity,has been known since the 1970's (Laird and Groman, J. Virol. 19:220,1976), and has been used in human clinical trials. See, U.S. Pat. No.5,792,458 and U.S. Pat. No. 5,208,021. As used herein, the term“diphtheria toxin” refers as appropriate to native diphtheria toxin orto diphtheria toxin that retains enzymatic activity but which has beenmodified to reduce non-specific toxicity.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). The term“ricin” also references toxic variants thereof. For example, see U.S.Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Ricinus communisagglutinin (RCA) occurs in two forms designated RCA₆₀ and RCA₁₂₀according to their molecular weights of approximately 65 and 120 kD,respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543,1972). The A chain is responsible for inactivating protein synthesis andkilling cells. The B chain binds ricin to cell-surface galactoseresidues and facilitates transport of the A chain into the cytosol(Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No. 3,060,165).

Ribonucleases have also been conjugated to targeting molecules for useas immunotoxins (see Suzuki et al., Nat Biotech 17:265-270, 1999).Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in,e.g., Rathore et al., Gene 190:31-35, 1997; and Goyal and Batra, Biochem345 Pt 2:247-254, 2000. Calicheamicins were first isolated fromMicromonospora echinospora and are members of the enediyne antitumorantibiotic family that cause double strand breaks in DNA that lead toapoptosis (see, e.g., Lee et al., J. Antibiot 42:1070-1087. 1989).

The drug is the toxic moiety of an immunotoxin in clinical trials (see,e.g., Gillespie et al., Ann Oncol 11:735-741, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B-chain (abrin-b)binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

In one embodiment, the toxin is Pseudomonas exotoxin (PE). NativePseudomonas exotoxin A (“PE”) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells. The native PE sequenceand the sequence of modified PE are provided in U.S. Pat. No. 5,602,095,incorporated herein by reference. In one embodiment, native PE has asequence set forth as:

(SEQ ID NO: 5) AEEAFDLWNE CAKACVLDLK DGVRSSRMSV DPAIADTNGQGVLHYSMVLE GGNDALKLAI DNALSITSDG LTIRLEGGVEPNKPVRYSYT RQARGSWSLN WLVPIGHEKP SNIKVFIHELNAGNQLSHMS PIYTIEMGDE LLAKLARDAT FFVRAHESNEMQPTLAISHA GVSVVMAQTQ PRREKRWSEW ASGKVLCLLDPLDGVYNYLA QQRCNLDDTW EGKIYRVLAG NPAKHDLDIKPTVISHRLHF PEGGSLAALT AHQACHLPLE TFTRHRQPRGWEQLEQCGYP VQRLVALYLAARLSWNQVDQ VIRNALASPGSGGDLGEAIR EQPEQARLAL TLAAAESERF VRQGTGNDEAGAANADVVSL TCPVAAGECA GPADSGDALL ERNYPTGAEFLGDGGDVSFS TRGTQNWTVE RLLQAHRQLE ERGYVFVGYHGTFLEAAQSI VFGGVRARSQ DLDAIWRGFY IAGDPALAYGYAQDQEPDAR GRIRNGALLR VYVPRSSLPG FYRTSLTLAAPEAAGEVERL IGHPLPLRLD AITGPEEEGG RLETILGWPLAERTVVIPSA IPTDPRNVGG DLDPSSIPDK EQAISALPDYASQPGKPPRE DLK

The method of action of PE is inactivation of the ADP-ribosylation ofelongation factor 2 (EF-2). The exotoxin contains three structuraldomains that act in concert to cause cytotoxicity. Domain Ia (aminoacids 1-252) mediates cell binding. Domain II (amino acids 253-364) isresponsible for translocation into the cytosol and domain III (aminoacids 400-613) mediates ADP ribosylation of elongation factor 2. Thefunction of domain Ib (amino acids 365-399) remains undefined, althougha large part of it, amino acids 365-380, can be deleted without loss ofcytotoxicity. See Siegall et al., J. Biol. Chem. 264:14256-14261, 1989.

The term “Pseudomonas exotoxin” (“PE”) as used herein refers asappropriate to a full-length native (naturally occurring) PE or to a PEthat has been modified. Such modifications may include, but are notlimited to, elimination of domain Ia, various amino acid deletions indomains Ib, II and III, single amino acid substitutions and the additionof one or more sequences at the carboxyl terminus, such as KDEL (SEQ IDNO: 6) and REDL (SEQ ID NO: 7) (see Siegall et al., supra). In severalexamples, the cytotoxic fragment of PE retains at least 50%, such asabout 75%, about 90%, or about 95% of the cytotoxicity of native PE. Inone embodiment, the cytotoxic fragment is more toxic than native PE.

Thus, the PE used in the immunotoxins disclosed herein includes thenative sequence, cytotoxic fragments of the native sequence, andconservatively modified variants of native PE and its cytotoxicfragments. Cytotoxic fragments of PE include those which are cytotoxicwith or without subsequent proteolytic or other processing in the targetcell (e.g., as a protein or pre-protein). Cytotoxic fragments of PEknown in the art include PE40, PE38, and PE35.

In several embodiments, the PE has been modified to reduce or eliminatenon-specific cell binding, typically by deleting domain Ia, as taught inU.S. Pat. No. 4,892,827, although this can also be achieved, forexample, by mutating certain residues of domain Ia. U.S. Pat. No.5,512,658, for instance, discloses that a mutated PE in which Domain Iais present but in which the basic residues of domain Ia at positions 57,246, 247, and 249 are replaced with acidic residues (glutamic acid, or“E”) exhibits greatly diminished non-specific cytotoxicity. This mutantform of PE is sometimes referred to as PE4E. PE40 is a truncatedderivative of PE (see, Pai et al., Proc. Nat'l Acad. Sci. U.S.A.88:3358-3362, 1991; and Kondo et al., J. Biol. Chem. 263:9470-9475,1988). PE35 is a 35 kD carboxyl-terminal fragment of PE in which aminoacid residues 1-279 have deleted and the molecule commences with a metat position 280 followed by amino acids 281-364 and 381-613 of nativePE. PE35 and PE40 are disclosed, for example, in U.S. Pat. No. 5,602,095and U.S. Pat. No. 4,892,827.

In some embodiments, the cytotoxic fragment PE38 is employed. PE38 is atruncated PE pro-protein composed of amino acids 253-364 and 381-613 ofSEQ ID NO: 3 which is activated to its cytotoxic form upon processingwithin a cell (see e.g., U.S. Pat. No. 5,608,039, and Pastan et al.,Biochim. Biophys. Acta 1333:C1-C6, 1997).

While in some embodiments, the PE is PE4E, PE40, or PE38, any form of PEin which non-specific cytotoxicity has been eliminated or reduced tolevels in which significant toxicity to non-targeted cells does notoccur can be used in the immunotoxins disclosed herein so long as itremains capable of translocation and EF-2 ribosylation in a targetedcell.

Conservatively modified variants of PE or cytotoxic fragments thereofhave at least about 80% sequence identity, such as at least about 85%sequence similarity, at least about 90% sequence identity, or at leastabout 95% sequence similarity at the amino acid level, with the PE ofinterest, such as PE38.

Nucleic Acids

Nucleic acids encoding antibodies that specifically bind GRP94, andconjugates and fusion thereof, are provided herein. These nucleic acidscan be used in conjunction with a BRAF inhibitor. With the antibodiesand immunotoxins herein provided, one of skill can readily construct avariety of clones containing functionally equivalent antibodies, andnucleic acids encoding these antibodies, such as nucleic acids whichdiffer in sequence but which encode the same effector molecule (“EM”) orantibody sequence. Thus, nucleic acids encoding antibodies andconjugates and fusion proteins are provided herein.

Nucleic acid sequences encoding the antibodies and/or immunotoxins canbe prepared by any suitable method including, for example, cloning ofappropriate sequences or by direct chemical synthesis by methods such asthe phosphotriester method of Narang, et al., Meth. Enzymol. 68:90-99,1979; the phosphodiester method of Brown et al., Meth. Enzymol.68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramiditetriester method described by Beaucage & Caruthers, Tetra. Letts.22(20):1859-1862, 1981, e.g., using an automated synthesizer asdescribed in, for example, Needham-VanDevanter et al. Nucl. Acids Res.12:6159-6168, 1984; and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

In one embodiment, the nucleic acid sequences encoding the antibody orimmunotoxin are prepared by cloning techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, an immunotoxin of use is prepared by inserting the cDNAwhich encodes a variable region into a vector which comprises the cDNAencoding the EM. The insertion is made so that the variable region andthe EM are read in frame so that one continuous polypeptide is produced.The polypeptide contains a functional Fv region and a functional EMregion. In one embodiment, cDNA encoding a cytotoxin is ligated to ascFv so that the cytotoxin is located at the carboxyl terminus of thescFv. In one example, cDNA encoding a Pseudomonas exotoxin (“PE”),mutated to eliminate or to reduce non-specific binding, is ligated to ascFv so that the toxin is located at the amino terminus of the scFv. Inanother example, PE38 is located at the amino terminus of the scFv. In afurther example, cDNA encoding a cytotoxin is ligated to a heavy chainvariable region of an antibody that binds the antigen of interest sothat the cytoxin is located at the carboxyl terminus of the heavy chainvariable region. The heavy chain-variable region can subsequently beligated to a light chain variable region of the antibody using disulfidebonds. In yet another example, cDNA encoding a cytotoxin is ligated to alight chain variable region of an antibody that binds the antigen (forexample, GRP94), so that the cytotoxin is located at the carboxylterminus of the light chain variable region. The light chain-variableregion can subsequently be ligated to a heavy chain variable region ofthe antibody using disulfide bonds.

Once the nucleic acids encoding the antibody immunotoxin is isolated andcloned, the protein can be expressed in a recombinantly engineered cellsuch as bacteria, plant, yeast, insect and mammalian cells. One or moreDNA sequences encoding an antibody immunotoxin can be expressed in vitroby DNA transfer into a suitable host cell. The cell may be prokaryoticor eukaryotic. The term also includes any progeny of the subject hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. Methods of stable transfer, meaning that the foreign DNA iscontinuously maintained in the host, are known in the art.

Polynucleotide sequences encoding the antibody or immunotoxin can beoperatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

The polynucleotide sequences encoding the antibody or immunotoxin can beinserted into an expression vector including, but not limited to aplasmid, virus or other vehicle that can be manipulated to allowinsertion or incorporation of sequences and can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the immunotoxin,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982). One of skill in the artcan readily use an expression systems such as plasmids and vectors ofuse in producing proteins in cells including higher eukaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide may becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the recombinantimmunotoxins can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially purecompositions of at least about 90 to 95% homogeneity are disclosedherein, and 98 to 99% or more homogeneity can be used for pharmaceuticalpurposes. Once purified, partially or to homogeneity as desired, if tobe used therapeutically, the polypeptides should be substantially freeof endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (e.g., 100-fold) ofthe denatured and reduced protein into refolding buffer. An exemplarybuffer is 0.1 M Tris, pH 8.0, 0.5 M 1-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. It isdesirable to add excess oxidized glutathione or other oxidizing lowmolecular weight compounds to the refolding solution after theredox-shuffling is completed.

In addition to recombinant methods, the immunoconjugates, EM, andantibodies disclosed herein can also be constructed in whole or in partusing standard peptide synthesis. Solid phase synthesis of thepolypeptides of less than about 50 amino acids in length can beaccomplished by attaching the C-terminal amino acid of the sequence toan insoluble support followed by sequential addition of the remainingamino acids in the sequence. Techniques for solid phase synthesis aredescribed by Barany & Merrifield, The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, andStewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are well known in the art.

BRAF Inhibitors

The methods disclosed herein utilize BRAF inhibitors. One exemplaryamino acid sequence for human BRAF is provided below.

MAALSGGGGG GAEPGQALFN GDMEPEAGAG AGAAASSAADPAIPEEVWNI KQMIKLTQEHIEALLDKFGG EHNPPSIYLEAYEEYTSKLD ALQQREQQLL ESLGNGTDFSVSSSASMDTVTSSSSSSLSV LPSSLSVFQN PTDVARSNPKSPQKPIVRVF LPNKQRTVVP ARCGVTVRDSLKKALMMRGLIPECCAVYRI QDGEKKPIGW DTDISWLTGE ELHVEVLENVPLTTHNFVRKTFFTLAFCDF CRKLLFQGFR CQTCGYKFHQRCSTEVPLMC VNYDQLDLLF VSKFFEHHPI PQEEASLAETALTSGSSPSA PASDSIGPQI LTSPSPSKSI PIPQPFRPADEDHRNQFGQRDRSSSAPNVH INTIEPVNID DLIRDQGFRGDGGSTTGLSA TPPASLPGSL TNVKALQKSPGPQRERKSSSSSEDRNRMKT LGRRDSSDDW EIPDGQITVG QRIGSGSFGT VYKGKWHGDVAVKMLNVTAPTPQQLQAFKN EVGVLRKTRH VNILLFMGYS TKPQLAIVTQWCEGSSLYHHLHIIETKFEM IKLIDIARQT AQGMDYLHAKSIIHRDLKSN NIFLHEDLTV KIGDFGLATV KSRWSGSHQFEQLSGSILWM APEVIRMQDK NPYSFQSDVY AFGIVLYELMTGQLPYSNINNRDQIIFMVG RGYLSPDLSK VRSNCPKAMKRLMAECLKKK RDERPLFPQI LASIELLARSLPKIHRSASEPSLNRAGFQT EDFSLYACAS PKTPIQAGGY GAFPVH (SEQ ID NO: 10, see GENBANK ®Accession No. ACD11489.1, incorporated herein by reference)

A number of BRAF inhibitors have been previously described (see, forexample, PCT Publication Nos. WO 2007/002325, WO 2007/002433, WO2009/047505, WO 03/086467; WO 2009/143024, WO 2010/104945, WO2010/104973, WO 2010/111527 and WO 2009/152087; U.S. Pat. Nos. 6,187,799and 7,329,670; and U.S. Patent Application Publication Nos. 2005/0176740and 2009/0286783, each of which is herein incorporated by reference).

PLX 4032 (also known as RG7204, RO5185426, and Vemurafenib,C₂₃H₁₈ClF₂N₃O₃S) is a BRAF small molecule inhibitor being developed byPlexxikon and Roche (Genentech) for the treatment of melanoma. Phase Iclinical trials in patients with advanced melanoma demonstrated that PLX4032 was effective in promoting tumor regression and increasing overallsurvival in patients with the V600E BRAF mutation. In particularembodiments of the present disclosure, the BRAF inhibitor is PLX 4032:

or a salt, solvate or functional derivative thereof. Thus, atherapeutically effective amount of PLX4032 can be used in combinationwith a therapeutically effective amount of an antibody that specificallybinds GRP94 (or a nucleic acid encoding this antibody) for the treatmentof tumors.

In other embodiments, the BRAF inhibitor is PLX 4720 (C₁₇H₁₄ClF₂N₃O₃S):

or a salt, solvate or functional derivative thereof. Thus, atherapeutically effective amount of PLX4720 can be used in combinationwith a therapeutically effective amount of an antibody that specificallybinds GRP94 (or a nucleic acid encoding this antibody) for the treatmentof tumors.

In further embodiments, the BRAF inhibitor is sofafenib(C₂₁H₁₆ClF₃N₄O₃):

or a salt, solvate or functional derivative thereof. Sofafenib (Nexavar)is used for the treatment of renal cancer, liver cancer, thyroid cancer,lung cancer, glioblastoma and kidney cancer. Thus a therapeuticallyeffective amount of sofafenib can be used in combination with atherapeutically effective amount of an antibody that specifically bindsGRP94 (or a nucleic acid encoding this antibody) for the treatment ofthese tumors.

In some embodiments, the BRAF inhibitors have the structure of FormulaIII, shown below, or any salts, prodrugs, tautomers or isomers thereof,as described in PCT Publication Nos. WO 2007/002325 and WO 2007/002433(which are incorporated herein by reference):

wherein: Q has a structure selected from the group consisting of

in which

indicates the attachment point of Q to A of Formula III;

-   Z₂ is N or CR¹²; Z₄ is N or CR¹⁴; Z₅ is N or CR¹⁵; Z₆ is N or CR¹⁶;-   L₂ is selected from the group consisting of    —(CR¹⁰R¹¹)_(p)—NR²⁵—(CR¹⁰R¹¹)_(q)—, —(CR¹⁰R¹¹)_(p)—O—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—S—(CR¹⁰R¹¹)_(q)—, —(CR¹⁰R¹¹)_(p)—C(O)—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—C(S)—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—S—(O)—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—S(O)₂—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—C(O)NR²⁵—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—C(S)NR²⁵—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—S(O)₃NR²⁵—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—NR²⁵C(O)—(CR¹⁰R¹¹)_(q)—,    —(CR¹⁰R¹¹)_(p)—NR²⁵C(S)—(CR¹⁰R¹¹)_(q)—, and    —(CR¹⁰R¹¹)_(p)—NR²⁵S(O)₂—(CR¹⁰R¹¹)_(q)—,-   p and q are independently 0, 1, or 2 provided, however, that at    least one of p and q is 0;-   s is 1 or 2;-   X is O or S;-   A is selected from the group consisting of —O—, —S—, —CR^(a)R^(b)—,    —NR¹—, —C(O)—, —C(S)—, —S(O)—, and —S(O)₂—;-   R^(a) and R^(b) at each occurrence are independently selected from    the group consisting of hydrogen, fluoro, —OH, —NH₂, lower alkyl,    lower alkoxy, lower alkylthio, mono-alkylamino, di-alkylamino, and    —NR⁸R⁹, wherein the alkyl chain(s) of lower alkyl, lower alkoxy,    lower alkylthio, mono-alkylamino, or di-alkylamino are optionally    substituted with one or more substituents selected from the group    consisting of fluoro, —OH, —NH₂, lower alkoxy, fluoro substituted    lower alkoxy, lower alkylthio, fluoro substituted lower alkylthio,    mono-alkylamino, di-alkylamino, and cycloalkylamino, provided,    however, that any substitution of the alkyl chain carbon bound to O    of alkoxy, S of thioalkyl or N of mono- or di-alkylamino is fluoro;    or-   R^(a) and R^(b) combine to form a 3-7 membered monocyclic cycloalkyl    or 5-7 membered monocyclic heterocycloalkyl, wherein the monocyclic    cycloalkyl or monocyclic heterocycloalkyl are optionally substituted    with one or more substituents selected from the group consisting of    halogen, —OH, —NH₂, lower alkyl, fluoro substituted lower alkyl,    lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,    fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino,    and cycloalkylamino;-   R¹ is selected from the group consisting of hydrogen, lower alkyl,    cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —C(O)R⁷, —C(S)R⁷,    —S(O)₂R⁷, —C(O)NHR⁷, —C(S)NHR⁷, and —S(O)₂NHR⁷, wherein lower alkyl    is optionally substituted with one or more substituents selected    from the group consisting of fluoro, —OH, —NH₂, lower alkoxy, lower    alkylthio, mono-alkylamino, di-alkylamino, and —NR⁸R⁹, wherein the    alkyl chain(s) of lower alkoxy, lower alkylthio, mono-alkylamino, or    di-alkylamino are optionally substituted with one or more    substituents selected from the group consisting of fluoro, —OH,    —NH₂, lower alkoxy, fluoro substituted lower alkoxy, lower    alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,    di-alkylamino, and cycloalkylamino, provided, however, that any    substitution of the alkyl chain carbon bound to O of alkoxy, S of    thioalkyl or N of mono- or di-alkylamino is fluoro, further provided    that when R¹ is lower alkyl, any substitution on the lower alkyl    carbon bound to the N of —NR¹— is fluoro, and wherein cycloalkyl,    heterocycloalkyl, aryl or heteroaryl are optionally substituted with    one or more substituents selected from the group consisting of    halogen, —OH, —NH₂, lower alkyl, fluoro substituted lower alkyl,    lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,    fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino,    and cycloalkylamino;-   R⁷ is selected from the group consisting of lower alkyl, cycloalkyl,    heterocycloalkyl, aryl, and heteroaryl, wherein lower alkyl is    optionally substituted with one or more substituents selected from    the group consisting of fluoro, —OH, —NH₂, lower alkoxy, lower    alkylthio, mono-alkylamino, di-alkylamino, and —NR⁸R⁹, provided,    however, that any substitution of the alkyl carbon bound to the N of    —C(O)NHR⁷, —C(S)NHR⁷ or —S(O)₂NHR⁷ is fluoro, wherein the alkyl    chain(s) of lower alkoxy, lower alkylthio, mono-alkylamino, or    di-alkylamino are optionally substituted with one or more    substituents selected from the group consisting of fluoro, —OH,    —NH₂, lower alkoxy, fluoro substituted lower alkoxy, lower    alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,    di-alkylamino, and cycloalkylamino, provided, however, that any    substitution of the alkyl chain carbon bound to O of alkoxy, S of    thioalkyl or N of mono- or di-alkylamino is fluoro, and wherein    cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally    substituted with one or more substituents selected from the group    consisting of halogen, —OH, —NH₂, lower alkyl, fluoro substituted    lower alkyl, lower alkoxy, fluoro substituted lower alkoxy, lower    alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,    di-alkylamino, and cycloalkylamino;-   R⁴, R⁵, R⁶, R¹², R¹⁴, R¹⁵, R¹⁶, R⁴², R⁴³, R⁴⁵, R⁴⁶, and R⁴⁷ are    independently selected from the group consisting of hydrogen,    halogen, optionally substituted lower alkyl, optionally substituted    lower alkenyl, optionally substituted lower alkynyl, optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl,    optionally substituted aryl, optionally substituted heteroaryl, —CN,    —NO₂, —CR^(a)R^(b)R²⁶, and -LR²⁶;-   L at each occurrence is independently selected from the group    consisting of -(alk)_(a)-S-(alk)_(b)-, -(alk)_(a)-O-(alk)_(b)-,    -(alk)_(a)-NR²⁵-(alk)_(b)-, -(alk)_(a)-C(O)-(alk)_(b)-,    -(alk)_(a)-C(S)-(alk)_(b)-, -(alk)_(a)-S(O)-(alk)_(b)-,    -(alk)_(a)-S(O)₂-(alk)_(b)-, -(alk)_(a)-OC(O)-(alk)_(b)-,    -(alk)_(a)-C(O)O-(alk)_(b)-, -(alk)_(a)-OC(S)-(alk)_(b)-,    -(alk)_(a)-C(S)O-(alk)_(b)-, -(alk)_(a)-C(O)NR²⁵-(alk)_(b)-,    -(alk)_(a)-C(S)NR²⁵-(alk)_(b)-, -(alk)_(a)-S(O)₂NR²⁵-(alk)_(b)-,    -(alk)_(a)-NR²⁵C(O)-(alk)_(b)-, -(alk)_(a)-NR²⁵C(S)-(alk)_(b)-,    -(alk)_(a)-NR²⁵S(O)₂-(alk)_(b)-, -(alk)_(a)-NR²⁵C(O)O-(alk)_(b)-,    -(alk)_(a)-NR²⁵C(S)O-(alk)_(b)-, -(alk)_(a)-OC(O)NR²⁵-(alk)_(b)-,    -(alk)_(a)-OC(S)NR²⁵-(alk)_(b)-, -(alk)_(a)-NR²⁵C(O)NR²⁵-(alk)_(b)-,    -(alk)_(a)-NR²⁵C(S)NR²⁵-(alk)_(b)-, and    -(alk)_(a)-NR²⁵S(O)₂NR²⁵-(alk)_(b)-;-   a and b are independently 0 or 1;-   alk is C₁₋₃ alkylene or C₁₋₃ alkylene substituted with one or more    substituents selected from the group consisting of fluoro, —OH,    —NH₂, lower alkyl, lower alkoxy, lower alkylthio, mono-alkylamino,    di-alkylamino, and —NR⁸R⁹, wherein lower alkyl or the alkyl chain(s)    of lower alkoxy, lower alkylthio, mono-alkylamino or di-alkylamino    are optionally substituted with one or more substituents selected    from the group consisting of fluoro, —OH, —NH₂, lower alkoxy, fluoro    substituted lower alkoxy, lower alkylthio, fluoro substituted lower    alkylthio, mono-alkylamino, di-alkylamino and cycloalkylamino,    provided, however, that any substitution of the alkyl chain carbon    bound to O of alkoxy, S of thioalkyl or N of mono- or di-alkylamino    is fluoro;-   R²⁵ at each occurrence is independently selected from the group    consisting of hydrogen, optionally substituted lower alkyl,    optionally substituted cycloalkyl, optionally substituted    heterocycloalkyl, optionally substituted aryl, and optionally    substituted heteroaryl;-   R²⁶ at each occurrence is independently selected from the group    consisting of hydrogen, provided, however, that hydrogen is not    bound to any of S(O), S(O)₂, C(O) or C(S) of L, optionally    substituted lower alkyl, optionally substituted lower alkenyl,    provided, however, that when R²⁶ is optionally substituted lower    alkenyl, no alkene carbon thereof is bound to N, S, O, S(O), S(O)₂,    C(O) or C(S) of L, optionally substituted lower alkynyl, provided,    however, that when R²⁶ is optionally substituted lower alkynyl, no    alkyne carbon thereof is bound to N, S, O, S(O), S(O)₂, C(O) or C(S)    of L, optionally substituted cycloalkyl, optionally substituted    heterocycloalkyl, optionally substituted aryl, and optionally    substituted heteroaryl;-   R¹⁰ and R¹¹ at each occurrence are independently selected from the    group consisting of hydrogen, fluoro, lower alkyl, and lower alkyl    optionally substituted with one or more substituents selected from    the group consisting of fluoro, —OH, —NH₂, lower alkoxy, fluoro    substituted lower alkoxy, lower alkylthio, fluoro substituted lower    alkylthio, mono-alkylamino, di-alkylamino, and cycloalkylamino; or-   any two of R¹⁰ and R¹¹ on the same or adjacent carbon atoms combine    to form a 3-7 membered monocyclic cycloalkyl or 5-7 membered    monocyclic heterocycloalkyl, and any others of R¹⁰ and R¹¹ are    independently selected from the group consisting of hydrogen,    fluoro, lower alkyl, and lower alkyl optionally substituted with one    or more substituents selected from the group consisting of fluoro,    —OH, —NH₂, lower alkoxy, fluoro substituted lower alkoxy, lower    alkylthio, fluoro substituted lower alkylthio, mono-alkylamino,    di-alkylamino, and cycloalkylamino, and wherein the monocyclic    cycloalkyl or monocyclic heterocycloalkyl are optionally substituted    with one or more substituents selected from the group consisting of    halogen, —OH, —NH₂, lower alkyl, fluoro substituted lower alkyl,    lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,    fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino,    and cycloalkylamino;-   R⁸ and R⁹ combine with the nitrogen to which they are attached to    form a 5-7 membered heterocycloalkyl optionally substituted with one    or more substituents selected from the group consisting of fluoro,    —OH, —NH₂, lower alkyl, fluoro substituted lower alkyl, lower    alkoxy, fluoro substituted lower alkoxy, lower alkylthio, and fluoro    substituted lower alkylthio;-   R¹⁷ is selected from the group consisting of hydrogen, halogen,    optionally substituted lower alkyl and —OR¹⁸;-   R³¹ and R³³ are independently selected from the group consisting of    optionally substituted aryl, optionally substituted heteroaryl,    optionally substituted cycloalkyl, and optionally substituted    heterocycloalkyl;-   R³⁶ is selected from the group consisting of substituted methyl,    optionally substituted C₂₋₆ alkyl, optionally substituted lower    alkenyl, provided, however, that when R³⁶ is optionally substituted    lower alkenyl, no alkene carbon thereof is bound to the S(O)₂ of    S(O)₂R³⁶, optionally substituted lower alkynyl, provided, however,    that when R³⁶ is optionally substituted lower alkynyl, no alkyne    carbon thereof is bound to the S(O)₂ of S(O)₂R³⁶, optionally    substituted cycloalkyl, optionally substituted heterocycloalkyl,    optionally substituted aryl, optionally substituted heteroaryl, and    —NR¹⁹R²⁰;-   R¹⁹, R²⁰, R³⁴, R³⁵, R³⁷, and R³⁸ are independently selected from the    group consisting of hydrogen, optionally substituted lower alkyl,    optionally substituted lower alkenyl, provided, however, that when    R¹⁹, R²⁰, R³⁴, R³⁵, R³⁷, or R³⁸ is optionally substituted lower    alkenyl, no alkene carbon thereof is bound to the N of NR¹⁹R²⁰,    NR³⁴R³⁵ or NR³⁷R³⁸, optionally substituted lower alkynyl, provided,    however, that when R¹⁹, R²⁰, R³⁴, R³⁵, R³⁷, or R³⁸ is optionally    substituted lower alkynyl, no alkyne carbon thereof is bound to the    N of NR¹⁹R²⁰, NR³⁴R³⁵ or NR³⁷R³⁸, optionally substituted cycloalkyl,    optionally substituted heterocycloalkyl, optionally substituted aryl    and optionally substituted heteroaryl; or-   R³⁴ and R³⁵ together with the nitrogen to which they are attached    form optionally substituted 5-7 membered heterocycloalkyl or    optionally substituted 5 or 7 membered nitrogen containing    heteroaryl; or-   R³⁷ and R³⁸ together with the nitrogen to which they are attached    form optionally substituted 5-7 membered heterocycloalkyl or    optionally substituted 5 or 7 membered nitrogen containing    heteroaryl;-   R³² is selected from the group consisting of hydrogen, optionally    substituted lower alkyl, optionally substituted cycloalkyl,    optionally substituted heterocycloalkyl, optionally substituted    aryl, optionally substituted heteroaryl, and —OR¹⁸;-   R⁸² is selected from hydrogen or lower alkyl; and-   R¹⁸ is hydrogen or optionally substituted lower alkyl;

In other embodiments of the present disclosure, the BRAF inhibitorcomprises a formula as described in PCT Publication No. WO 03/086467(incorporated herein by reference) as Formula I, Formula II or FormulaIII:

Formula I—

or a salt, solvate, physiologically functional derivative thereof;wherein

Y is CR¹ and V is N; or Y is CR¹ and V is CR²;

R¹ represents a group CH₃SO₂CH₂CH₂NHCH₂—Ar—, wherein Ar is selected fromphenyl, furan, thiophene, pyrrole and thiazole, each of which mayoptionally be substituted by one or two halo, C₁₋₄ alkyl or C₁₋₄ alkoxygroups;R² is selected from the group comprising hydrogen, halo, hydroxy, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylamino and di[C_(1-4 alkyl)]amino;U represents a phenyl, pyridyl, 3H-imidazolyl, indolyl, isoindolyl,indolinyl, isoindolinyl, 1H-indazolyl, 2,3-dihydro-1H-indazolyl,1H-benzimidazolyl, 2,3-dihydro-1H-benzimidazolyl or 1H-benzotriazolylgroup, substituted by an R³ group and optionally substituted by at leastone independently selected R⁴ group;R³ is selected from a group comprising benzyl, halo-, dihalo- andtrihalobenzyl, benzoyl, pyridylmethyl, pyridylmethoxy, phenoxy,benzyloxy, halo-, dihalo- and trihalobenzyloxy and benzenesulphonyl;or R³ represents trihalomethylbenzyl or trihalomethylbenzyloxy;or R³ represents a group of formula

wherein each R⁵ is independently selected from halogen, C₁₋₄ alkyl andC₁₋₄ alkoxy; and n is 0 to 3;each R⁴ is independently hydroxy, halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl,C₂₋₄ alkynyl, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino, di[C₁₋₄ alkyl]amino,C₁₋₄ alkylthio, C₁₋₄ alkylsulphinyl, C₁₋₄ alkylsulphonyl, C₁₋₄alkylcarbonyl, carboxy, carbamoyl, C₁₋₄ alkoxycarbonyl, C₁₋₄alkanoylamino, N—(C₁₋₄ alkyl)carbamoyl, N,N-di(C₁₋₄ alkyl) carbamoyl,cyano, nitro and trifluoromethyl.

Additional BRAF inhibitors are shown below:

wherein R is —Cl or —Br, X is CH, N, or CF, and Z is thiazole or furan.

In some embodiments, the BRAF inhibitor is selected from a compound asdescribed in PCT Publication No. WO 2010/104973 which is incorporated byreference herein. These include the following:

In a first aspect, a compound selected from the group consisting ofN-[3-(4-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-4-trifluorometh)/1-benzenesulfonamide(P-0001),N-[3-(4-ethynyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-4-trifluoromethyl-benzenesulfonamide(P-0002), any salt thereof, any formulation thereof, any conjugatethereof, any derivative thereof, and any form thereof is provided. Incertain embodiments P-0001, P-0002, or a salt thereof, formulationthereof, conjugate thereof, derivative thereof, or form thereof is aninhibitor of one or more Raf protein kinases, including A-Raf, B-Raf,and c-Raf-1 (including any mutations of these kinases),

In a second aspect the compoundN-[3-(4-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-4-trifluoromethyl-benzenesulfonamide(P-0001), or a salt thereof, formulation thereof, conjugate thereof,derivative thereof, or form thereof is provided. In certain embodimentsP-0001, or a salt thereof, formulation thereof, conjugate thereof,derivative thereof, or form thereof is an inhibitor of one or more Rafprotein kinases, including

A-Raf, B-Raf, and c-Raf-1 (including any mutations of these kinases).

In a third aspect the compoundN-[3-(4-ethynyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-4-trifluoromethyl-benzenesulfonamide(P-0002), or a salt thereof, formulation thereof, conjugate thereof,derivative thereof, or form thereof is provided. In certain embodimentsP-0002, or a salt thereof, formulation thereof, conjugate thereof,derivative thereof, or form thereof is an inhibitor of one or more Rafprotein kinases, including A-Raf, B-Raf, and c-Raf-1 (including anymutations of these kinases).

In some embodiments, the BRAF inhibitor is selected from a compound asdescribed in PCT Publication No. WO 2010/104945, which is incorporatedby reference herein. These include the following:

In a first aspect, a compound selected from the group consisting ofpropane-1-sulfonic acid{2,4-difluoro-3-[5-(2-methoxy-pyrimidin-5-yl)-1H-pyr[tau]olo[2,3-bJpyridinc-3-carbonyl]-phenyl}-amide(P-0001), propane-1-sulfonic acid[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenylj-amide(P-0002), propane-1-sulfonic acid[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2-fluoro-phenyl]-amide(P-0003),N-[3-(5-cyano-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-2,5-diflu[upsilon]r[upsilon]-ben/enesulfonamide(P-0004),N-[3-(5-cyano-1H-pyrrolo[2,3-b[pyridine-3-carbonyl)-2,4-difluoro-phenylJ-3-fluoro-benzenesulfonamide(P-0005), pyrrolidine-1-sulfonic acid[3-(5-cyano-1H-pyi[tau]olo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-amide(P-0006), N,N-dimethylamino-sulfonic acid[3-(5-cyano-1H-pyrrolo[2,3-bJpyridine-3-carbonyl)-2.4-difluoro-phenylJ-amide(P-0007), any salt thereof, any formulation thereof, any conjugatethereof, any derivative thereof, and any form thereof is provided. Incertain embodiments P-0001, P-0002, P-0003, P-0004, P-0005, P-0006,P-0007. or a salt thereof, formulation thereof, conjugate thereof,derivative thereof, or form thereof is an inhibitor of one or more Rafprolei[pi]kinases, including [Lambda]-Raf, R-Raf. and c-Raf-1 (includingany mutations of these kinases).

In some embodiments, the BRAF inhibitor is a compound having a formulafrom U.S. Pat. No. 6,187,799, which is incorporated by reference herein:

In some embodiments, the BRAF inhibitor is a compound as disclosed inPCT Publication No. WO 2010/111527, which is incorporated by referenceherein. For example, the BRAF inhibitor can have a structure accordingto Formula I of PCT Publication No. WO 2010/111527.

or a salt, a prodrug, a tautomer or an isomer thereof,

wherein:

Ar is selected from the group consisting of:

wherein

indicates the point of attachment of Ar to Lj of Formula I and

indicates the point of attachment of Ar to L̂ of Formula I;

L, is selected from the group consisting Of —C(R⁵R⁶)—, —C(O)—, —C(S)—,—N(R⁷)—, —O—, —S—, —S(O)—, and —S(O)2-;

L₂ is selected from the group consisting of —N(R⁸)—C(O)—, —N(R⁸)—C(S)—,—N(R⁸)—S(O)—N(R⁸)—S(O)2-, —N(R⁸)—C(O)—N(R⁸)—, —N(R⁸)—C(S)—N(R⁸)—, and—N(R⁸)—S(O)2-N(R⁸)—;

R¹ is selected from the group consisting of optionally substituted loweralkyl, optionally substituted lower alkenyl, optionally substitutedlower alkynyl, optionally substituted cycloalkyl, optionally substitutedhetcrocycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl;

R² is selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted lower alkynyl, optionally substitutedcycloalkyl, optionally substituted hetcrocycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —CN, —NO₂, —O—R⁹,—S—R¹¹, —N(R⁹)—R¹⁰, —C(O)—R¹¹, —C(S)—R¹¹, —C(O)—N(R⁹R¹⁰,—C(S)—N(R⁹)—R¹⁰, —C(O)—N(R¹³)—OR⁹, —C(S)—N(R¹³)—OR⁹,—C(O)—N(R¹³)—S(O)₂—R¹¹, —C(S)—N(R¹³)—S(O)₂—R¹¹, —C(O)—O—R⁹, —S(O)—R¹¹,—S(O)₂—R¹¹, —S(O)—N(R⁹)—R¹⁰, —S(O)₂—N(R⁹)—R¹⁰, —S(O)₂—N(R¹³)—C(O)R¹¹,—S(O)₂—N(R¹³)—C(S)R¹¹, —N(R¹³)—C(O)—R¹¹, —N(R¹³)—C(S)—R¹¹,—N(R¹³)—S(O)—R¹¹, —N(R¹³)—S(O)₂—R¹¹, —N(R¹³)—C(O)—N(R⁹)—R¹⁰,—N(R¹³)—C(S)—N(R⁹)—R¹⁰, and —N(R¹³)—S(O)₂—N(R⁹)—R¹⁰;

R³ is selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted lower alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —CN, —NO₂, —O—R¹⁷,—S—R¹⁹, —N(R¹⁷)—R¹⁸, —C(O)—R¹⁹, —C(S)—R¹⁹, —C(O)—N(R¹⁷R¹⁸,—C(S)—N(R¹⁷)—R¹⁸, —C(O)—N(R²⁰)—OR¹⁷, —C(S)—N(R²⁰)—OR¹⁷,—C(O)—N(R²⁰)—S(O)₂—R¹⁹, —C(S)—N(R²⁰)—S(O)₂—R¹⁹, —C(O)—O—R¹⁷, —S(O)—R¹⁹,—S(O)₂—R¹⁹, —S(O)—N(R¹⁷)—R¹⁸, —S(O)₂—N(R¹⁷)—R¹⁸, —S(O)₂—N(R²⁰)—C(O)R¹⁹,—S(O)₂—N(R²⁰)—C(S)R¹⁹, —N(R²⁰)—C(O)—R¹⁹, —N(R²⁰)—C(S)—R¹⁹,—N(R²⁰S(O)—R¹⁹, —N(R²⁰S(O)₂—R¹⁹, —N(R²⁰C(O)—N(R¹⁷)—R¹⁸,—N(R²⁰)—C(S)—N(R¹⁷)—R¹⁸, and —N(R²⁰)—S(O)₂—N(R¹⁷)—R¹⁸;

R⁴ is selected from the group consisting of hydrogen, halogen,optionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted lower alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —CN, NO₂, —O—R²¹,—S—R²³, —N(R²¹)—R²², —C(O)—R²³, —C(S)—R²³, —C(O)—N(R²¹)—R²²,—C(S)—N(R²¹)—R²², —C(O)—N(R²⁴)—OR²¹, —C(S)—N(R²⁴)—OR²¹,—C(O)—N(R²⁴)—S(O)₂—R²³, —C(S)—N(R²⁴)—S(O)₂—R²³, —C(O)—O—R²¹, —S(O)₂—R²³,—S(O)₂—R²³, —S(O)—N(R²¹)—R²², —S(O)2-N(R²¹)—R²², —S(O)₂—N(R²⁴)—C(O)R²³,—S(O₂—N(R²⁴)—C(S)R²³, —N(R²⁴)—C(O)—R²³, —N(R²⁴)—C(S)—R²³,—N(R²⁴)—S(O)—R²³, —N(R²⁴)—S(O)₂—R²³, —N(R²⁴)—C(O)—N(R²¹)—R²²,—N(R²⁴)—C(S)—N(R²¹)—R²², and —N(R²⁴)—S(O)₂—N(R²¹)—R²²;

R⁵ and R⁶ are independently selected from the group consisting ofhydrogen, fluoro, —OH, —NH₂, lower alkyl, lower alkoxy, loweralklylthio, mono-alkylamino, di-alkylamino, and —N(R²⁵)—R²⁶, wherein thealkyl chain(s) of lower alkyl, lower alkoxy, lower alkylthio,mono-alkylamino, or di-alkylamino are optionally substituted with one ormore substituents selected from the group consisting of fluoro, —OH,—NH₂, lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,fluoro substituted lower alkylthio, mono-alkylamino, di-alkylamino, andcycloalkylamino; or

R⁵ and R⁶ combine to form a 3-7 membered monocyclic cycloalkyl or 5-7membered monocyclic heterocycloalkyl, wherein the 3-7 memberedmonocyclic cycloalkyl or 5-7 membered monocyclic heterocycloalkyl areoptionally substituted with one or more substituents selected from thegroup consisting of halogen, —OH, —NH₂, lower alkyl, fluoro substitutedlower alkyl, lower alkoxy, fluoro substituted lower alkoxy, loweralkylthio, fluoro substituted lower alkylthio, mono-alkylamino,di-alkylamino, and cycloalkylamino,

R⁷, R¹³, R²⁰, and R²⁴ are independently selected from the groupconsisting of hydrogen, optionally substituted lower alkyl, optionallysubstituted cycloalkyl, optionally substituted heterocycloalkyl,optionally substituted aryl, optionally substituted heteroaryl,—C(O)—R²⁷, —C(S)—R²⁷, —S(O)—R²⁷, —S(O)₂—R²⁷, —C(O)—N(H)—R²⁷,—C(S)—N(H)—R²⁷, and —S(O)₂—N(H)—R²⁷,

R⁸ at each occurrence is independently hydrogen, lower alkyl, or loweralkyl substituted with one or more substituents selected from the groupconsisting of fluoro, —OH, —NH₂, lower alkoxy, fluoro substituted loweralkoxy, lower alkylthio, fluoro substituted lower alkylthio,mono-alkylamino, fluoro substituted mono-alkylamino, di-alkylamino,fluoro substituted di-alkylamino, and —N(R²⁵)—R²⁶,

R¹², R¹⁴, R¹⁵, and R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, optionally substituted lower alkyl,—N(R²⁸)—R²⁹, —O—R²⁸, and —S—R³⁰,

R¹¹, R¹⁹ and R²³ are independently selected from the group consisting ofoptionally substituted lower alkyl, optionally substituted loweralkenyl, optionally substituted lower alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl,

R⁹, R¹⁰, R¹⁷, R¹⁸, R²¹ and R²² are independently selected from the groupconsisting of hydrogen, optionally substituted lower alkyl, optionallysubstituted lower alkenyl, optionally substituted lower alkynyl,optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl;

R²⁵ and R²⁶ at each occurrence combine with the nitrogen to which theyare attached to form a 5-7 membered heterocycloalkyl optionallysubstituted with one or more substituents selected from the groupconsisting of fluoro, —OH, —NH₂, lower alkyl, fluoro substituted loweralkyl, lower alkoxy, fluoro substituted lower alkoxy, lower alkylthio,and fluoro substituted lower alkylthio;

R²⁷ at each occurrence is independently selected from the groupconsisting of optionally substituted lower alkyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl;

R²⁸ and R²⁹ at each occurrence are independently hydrogen or optionallysubstituted lower alkyl; and

R³⁰ at each occurrence is optionally substituted lower alkyl.

In some embodiments, the BRAF inhibitor is a compound having the formuladisclosed in U.S. Pat. No. 7,329,670, which is incorporated by referenceherein. For example, the BRAF inhibitor can have the following structure

wherein B is generally an unsubstituted or substituted, up to tricyclic,aryl or heteroaryl moiety with up 30 carbon atoms with at least one 5 or6 member aromatic structure containing 0-4 members of the groupconsisting of nitrogen, oxygen and sulfur. A is a heteroaryl moiety.

In some examples of the presence disclosure, the BRAF inhibitor is GDC0789 (Genentech):

In other examples, the inhibitor is PD-325901, XL518, PD-184352,PD-318088, AZD6244 or CI-1040 (see PCT Publication No. WO 2009/047505,incorporated herein by reference).

In further examples, the BRAF inhibitor can be an antibody thatspecifically binds BRAF, or an antigen binding fragment thereof. Thisincludes monoclonal antibodies and antigen binding fragments thatspecifically bind BRAF. The BRAF inhibitor can also be an inhibitoryRNA, such as, but not limited to, anti-sense RNA, small inhibitor RNA,and shRNA.

Methods of Treatment

Methods are provided herein for treating a subject diagnosed with atumor that expresses a mutated BRAF. The methods include administeringto the subject (1) a therapeutically effective amount of an antibody orantigen binding fragment thereof that specifically binds glucoseregulated protein (GRP) 94, or a nucleic acid encoding the antibody orantigen binding fragment; and (2) a therapeutically effective amount ofa BRAF inhibitor, thereby treating the cancer in the subject.

These use of a BRAF inhibitor in combination with an antibody or antigenbinding fragment thereof that specifically binds GRP94 provide anunexpectedly superior result for the treatment of any tumor, whereincells in the tumor comprise a BRAF mutation. In other embodiments, theBRAF mutation is a V600E mutation. In other embodiments, the BRAFmutation is R462I, I463S, G464E, G464V, G466A, G466E, G466V, G469A,G469E, N581S, E585K, D594V, F595L, G596R, L597V, T599I, V600D, V600E,V600K, V600R, K601E, and A728V. In further embodiments the BRAF mutationis in one of two regions: the glycine-rich P loop of the N lobe and theactivation segment and flanking regions. The cells in the tumor can havemore than one BRAF mutation.

In additional embodiments, the subject has a primary or secondaryresistance to the BRAF inhibitor. A subject that does not respondinitially to a BRAF inhibitor has primary resistance. A subject thatinitially responds to the BRAF inhibitor, but then ceases to respond,has secondary resistance. In some examples, the subject responds forone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve months, but then does not respond to the BRAF inhibitor. Thus,the methods can include selecting a subject with a resistance to a BRAFinhibitor, such as selecting a subject with primary resistance to theBRAF inhibitor or secondary resistance to the BRAF inhibitor.

The subject can have a tumor. Thus, in some embodiments, the methodincludes selecting a subject that has secondary resistance to a BRAFinhibitor, such as a subject with secondary resistance to a BRAFinhibitor that has a tumor, such as melanoma. In other embodiments, themethod includes selecting a subject with primary resistance to a BRAFinhibitor, such as a subject with primary resistance to a BRAF inhibitorthat has a tumor, such as melanoma.

The disclosed methods can also be used to prevent metastasis or decreasethe number of micrometastases, such as micrometastases to regional lymphnodes. Disclosed herein are methods to treat a subject diagnosed with atumor, such as a melanoma. Melanoma includes spreading melanoma, nodularmelanoma, acral lentiginous melanoma, and lentigo maligna (melanoma).However, the methods disclosed herein can also be used to treat othercancers, such breast cancer, prostate cancer, ovarian cancer, coloncancer, stomach cancer, pancreatic cancer, glioma, chordoma,chondrosarcoma, thyroid cancer, colon cancer, glioma or a squamous cellcarcinoma. Squamous cells carcinomas include, but are not limited tohead and neck squamous cell carcinoma, and squamous cell cancers of theskin, lung, prostate, esophagus, vagina and cervix. The methodsdisclosed herein can also be used to breast cancer, head and necksquamous cell carcinoma, renal cancer, lung cancer, glioma, bladdercancer, ovarian cancer, colon cancer or pancreatic cancer, wherein cellsof the cancer express GRP94. The methods also can be used to treatnon-Hodgkin lymphoma, colorectal cancer, papillary thyroid carcinoma,non-small cell lung carcinoma, and adenocarcinoma of lung. Thus, themethods can include selecting a subject with a tumor.

A variety of different types of melanoma may be treated in accordancewith the present methods including, such as superficial spreadingmelanoma, nodular malignant melanoma, acral lentiginous melanoma,lentiginous malignant melanoma, and mucosal lentiginous melanoma. Theprimary melanoma may also be cutaneous or extracutaneous. Extracutaneousprimary malignant melanomas include ocular melanoma and clear-cellsarcoma of the soft tissues. Additional indications include raremelanomas or precancerous lesions where relevance of RTK targets may beimplicated. The present methods are also useful in the treatment ofmelanoma that has metastasized.

A therapeutically effective amount of the agents will depend upon theseverity of the disease and the general state of the patient's health. Atherapeutically effective amount of the antibodies or antigen bindingfragments thereof (or nucleic acids encoding the antibody or antigenbinding fragment) and BRAF inhibitor is that which provides either areduction in tumor burden, decreased metastatic lesions and/orsubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

The antibody or antigen binding fragment can be administered in the sameformulation or separately. They can be given at the same time or adifferent time (simultaneously or sequentially), but sufficientlyconcurrently to have the beneficial effect disclosed herein.Compositions are provided herein that include a carrier and one or moreof the antibodies that specifically bind GRP94 and/or antigen bindingfragments thereof, in combination with a BRAF inhibitor. Compositionscomprising immunoconjugates or immunotoxins of these antibodies are alsoprovided. The compositions can be prepared in unit dosage forms foradministration to a subject. The amount and timing of administration areat the discretion of the treating physician to achieve the desiredpurposes.

The antibody (or a nucleic acid encoding the antibody) and/or BRAFinhibitor can be formulated for systemic or local (such as intra-tumor)administration. In one example, the antibodies and/or BRAF inhibitor isformulated for parenteral administration, such as intravenousadministration. In another example, the antibodies, antigen bindingfragments, nucleic acid and/or one or more BRAF inhibitors areformulated for topical administration, subcutaneous or intradermaladministration. A BRAF inhibitor and/or and antibody or antigen bindingfragment described above (or a nucleic acid encoding the antibody orantigen binding fragment) can be delivered transdermally via, forexample, a transdermal delivery device or a suitable vehicle or, such inan ointment base, which may be incorporated into a patch for controlleddelivery. Such devices are advantageous, as they may allow a prolongedperiod of treatment relative to, for example, an oral or intravenousmedicament. Examples of transdermal delivery devices may include, forexample, a patch, dressing, bandage or plaster adapted to release acompound or substance through the skin of a patient.

The compositions for administration can include a solution of theantibodies that specifically bind GRP94 (or nucleic acids encoding theseantibodies) and/or BRAF inhibitor(s) dissolved or suspended in apharmaceutically acceptable carrier, such as an aqueous carrier. Avariety of aqueous carriers can be used, for example, buffered salineand the like. These solutions are sterile and generally free ofundesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies can be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

Compositions for administering BRAF inhibitors are known in the art.Suitable BRAF inhibitors and pharmaceutical formations including theseinhibitors are disclosed for example, in PCT Publication No.WO2009047505A2, PCT Publication No. WO03086467A1, U.S. Published PatentApplication US20050176740A1, U.S. Pat. No. 6,187,799, U.S. Pat. No.7,329,670, PCT Publication No. WO2009143024A2, PCT Publication No.WO2010104945A1, PCT Publication No. WO2010104973A1, PCT Publication No.WO2010111527A1, U.S. Published Patent Application No. 20090286783A1 andPCT Publication No. WO2009152087A1, all of which are incorporated byreference herein.

The antibody or antigen binding fragment thereof that specifically bindsGRP94 (or a nucleic acid encoding the antibody or antigen bindingfragment) and the BRAF inhibitors can be administered to slow or inhibitthe growth of cells, such as cancer cells. In these applications, atherapeutically effective amount of an antibody is administered to asubject in an amount sufficient to inhibit growth, replication ormetastasis of cancer cells, or to inhibit a sign or a symptom of thecancer. In some embodiments, the compositions are administered to asubject to inhibit or prevent the development of metastasis, or todecrease the size or number of metasases, such as micrometastases, forexample micrometastases to the regional lymph nodes (Goto et al., Clin.Cancer Res. 14(11):3401-3407, 2008).

These compositions disclosed herein can be administered in conjunctionwith another chemotherapeutic agent, either simultaneously orsequentially. Many chemotherapeutic agents are presently known in theart. In one embodiment, the chemotherapeutic agents is selected from thegroup consisting of mitotic inhibitors, alkylating agents,anti-metabolites, intercalating antibiotics, growth factor inhibitors,cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survivalagents, biological response modifiers, anti-hormones, e.g.anti-androgens, and anti-angiogenesis agents.

Methods of treating melanoma can further include administering one ormore additional anti-cancer drugs for the treatment of melanoma with acompound as defined herein. For example, anti-cancer drugs for thetreatment of melanoma, especially metastatic melanoma, may be selectedfrom alkylating anti-cancer drugs such as dacarbazine, temozolomide,mechlorethamine, and nitrosoureas such as carmustine, lomustine, andfotemustine; taxanes, such as paclitaxel and docetaxel; vinca alkaloids,such as vinblastine; topoisomerase inhibitors such as irinotecan;thalidomide; anti-cancer antibiotics such as streptozocin anddactinomycin; or platinum anti-cancer drugs, such as cisplatin andcarboplatin. Compounds of the invention may be added topolychemotherapeutic regimes such as the Dartmouth regime, CVD(cisplatin, vinblastine, and dacarbazine) and BOLD (bleomycin,vincristine, lomustine, and dacarbazine). In some embodiments, theanti-cancer drugs are selected from interferons such as, but not limitedto, interferon alpha-2a, interferon alpha-2b, pegylated interferons suchas pegylated interferon alpha-2b. Interleukins such as interleukin-2 mayalso be used in combination with compounds disclosed herein.

In the methods of treating melanoma described herein, thetherapeutically effective amount of the compound can range from about0.25 mg/kg to about 30 mg/kg body weight of the subject. In someembodiments, the therapeutically effective amount of the compound canrange from about 0.5 mg/kg to about 30 mg/kg, from about 1 mg/kg toabout 30 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 1 mg/kgto about 15 mg/kg, or from about 1 or 2 mg/kg to about 10 mg/kg. Inother embodiments, the amount of the compound administered to thesubject ranges from about 25 to about 1500 mg/day and, preferably, fromabout 100 or 200 mg/day to about 500 or 600 mg/day.

Treatment may also include administering the pharmaceutical formulationsof the present methods in combination with other therapies. For example,a pharmaceutical formulations including the antibody and BRAF inhibitormay be administered before, during, or after a surgical procedure and/orradiation therapy.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with theantibody and the BRAF inhibitor. Examples of useful COX-II inhibitorsinclude CELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples ofuseful matrix metalloproteinase inhibitors are described in PCTPublication No. WO 96/33172 (published Oct. 24, 1996), PCT PublicationNo. WO 96/27583 (published Mar. 7, 1996), European Patent ApplicationNo. 97304971.1 (filed Jul. 8, 1997), European Patent Application No.99308617.2 (filed Oct. 29, 1999), PCT Publication No. WO 98/07697(published Feb. 26, 1998), PCT Publication No WO 98/03516 (publishedJan. 29, 1998), PCT Publication No WO 98/34918 (published Aug. 13,1998), PCT Publication No WO 98/34915 (published Aug. 13, 1998), PCTPublication No WO 98/33768 (published Aug. 6, 1998), PCT Publication NoWO 98/30566 (published Jul. 16, 1998), European Patent Publication606,046 (published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), PCT Publication No WO 90/05719 (published May31, 1990), PCT Publication No WO 99/52910 (published Oct. 21, 1999), PCTPublication No WO 99/52889 (published Oct. 21, 1999), PCT Publication NoWO 99/29667 (published Jun. 17, 1999), PCT International Application No.PCT/IB98/01113 (filed Jul. 21, 1998), European Patent Application No.99302232.1 (filed Mar. 25, 1999), U.S. Pat. No. 5,863,949 (issued Jan.26, 1999), U.S. Pat. No. 5,861,510 (issued Jan. 19, 1999), and EuropeanPatent Publication 780,386 (published Jun. 25, 1997). In one example,the MMP inhibitors do not induce arthralgia upon administration. Inanother example, the MMP inhibitor selectively inhibits MMP-2 and/orMMP-9 relative to the other matrix-metalloproteinases (such as MMP-1,MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, andMMP-13). Some specific examples of MMP inhibitors of use are AG-3340, RO32-3555, RS 13-0830,3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino-]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino-1-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxaicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino-1-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

The antibody or antigen binding fragment that specifically bind GRP94(or nucleic acid encoding the antibody or antigen binding fragment) andthe BRAF inhibitor can also be used with signal transduction inhibitors,such as agents that can inhibit EGF-R (epidermal growth factor receptor)responses, such as EGF-R antibodies, EGF antibodies, and molecules thatare EGF-R inhibitors; VEGF (vascular endothelial growth factor)inhibitors, such as VEGF receptors and molecules that can inhibit VEGF;and erbB2 receptor inhibitors, such as organic molecules or antibodiesthat bind to the erbB2 receptor, for example, HERCEPTIN™ (Genentech,Inc.). EGF-R inhibitors are described in, for example in PCT PublicationNos. WO 95/19970 (published Jul. 27, 1995), WO 98/14451 (published Apr.9, 1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Pat. No.5,747,498 (issued May 5, 1998). EGFR-inhibiting agents also include, butare not limited to, the monoclonal antibodies C225 and anti-EGFR 22Mab(ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200(Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. andMerck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787(Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033(Warner Lambert Parke Davis), CI-1033/PD 183,805 (Warner Lambert ParkeDavis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer MannheimGmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia),BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co.), VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864(INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker HughesCancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-RVaccine (York Medical/Centro de Immunologia Molecular (CIM)).

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be used in conjunction withan antibody that specifically binds endoplasmin. VEGF inhibitors aredescribed in, for example in PCT Publication No. WO 99/24440 (publishedMay 20, 1999), PCT International Application PCT/IB99/00797 (filed May3, 1999), PCT Publication No. WO 95/21613 (published Aug. 17, 1995), PCTPublication No. WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No.5,834,504 (issued Nov. 10, 1998), PCT Publication No. WO 98/50356(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), PCT Publication No. WO 99/10349(published Mar. 4, 1999), PCT Publication No. WO 97/32856 (publishedSep. 12, 1997), PCT Publication No. WO 97/22596 (published Jun. 26,1997), PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCTPublication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and PCT Publication No. WO 98/02437 (publishedJan. 22, 1998). Other examples of some specific VEGF inhibitors areIM862 (Cytran Inc.); anti-VEGF monoclonal antibody of Genentech, Inc.;and angiozyme, a synthetic ribozyme from Ribozyme and Chiron. These andother VEGF inhibitors can be used in conjunction with the antibodies,antigen binding fragments, and the BRAF inhibitor.

ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome plc), andthe monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), can furthermore be combined with the antibodies, antigenbinding fragments and BRAF inhibitor, for example those indicated in PCTPublication No. WO 98/02434 (published Jan. 22, 1998), PCT PublicationNo. WO 99/35146 (published Jul. 15, 1999), PCT Publication No. WO99/35132 (published Jul. 15, 1999), PCT Publication No. WO 98/02437(published Jan. 22, 1998), PCT Publication No. WO 97/13760 (publishedApr. 17, 1997), PCT Publication No. WO 95/19970 (published Jul. 27,1995), U.S. Pat. No. 5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No.5,877,305 (issued Mar. 2, 1999). ErbB2 receptor inhibitors of use arealso described in U.S. Provisional Application No. 60/117,341, filedJan. 27, 1999, and in U.S. Provisional Application No. 60/117,346, filedJan. 27, 1999.

For the treatment of cancer, such as melanoma, the antibody or antigenbinding fragment that specifically bind GRP94 (or nucleic acid encodingthe antibody or antigen binding fragment) and a BRAF inhibitor can beused with surgical treatment, or with another therapeutic includingdacarbazine (also termed DTIC), or interleukin-2 (IL-2) or interferon,such as interferon (IFN). For the treatment of a superficial melanoma,the antibody or antigen binding fragment that specifically bind GRP94(or nucleic acid encoding the antibody or antigen binding fragment) anda BRAF inhibitor can be used in conjunction with Imiquimod. However, forthe treatment of another cancer, such as head and neck squamous cellcarcinoma, the antibody or antigen binding fragment that specificallybind GRP94 (or nucleic acid encoding the antibody or antigen bindingfragment) and a BRAF inhibitor can be used in conjunction with surgery,radiation therapy, chemotherapy, other antibodies (such as cetuximab andbevacizumab) or small-molecule therapeutics (such as erlotinib). One ofskill in the art can readily determine surgical procedures andadditional chemotherapeutic agents of use.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies (or antigen binding fragmentsthereof) and BRAF inhibitor to effectively treat the patient. The dosagecan be administered once but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. The subject can be treated at regularintervals, such as daily, weekly, biweekly, or monthly, until a desiredtherapeutic result is achieved.

Generally, the dose is sufficient to treat or ameliorate symptoms orsigns of disease without producing unacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein, such as a cytotoxin or a drug, as a central core.In microspheres the therapeutic is dispersed throughout the particle.Particles, microspheres, and microcapsules smaller than about 1 μm aregenerally referred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and non-degradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm.

Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic molecules are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

The treatment of tumors, such as metastatic melanoma is changing rapidlydue to the great success in translational research from bench tobedside. PLX4032 (RG7204, Plexxikon,) is a selective V600E mutated BRAFinhibitor. A response rate of 70% in 32 BRAF mutated advanced melanomapatients treated by PLX4032 was reported in the phase II study (Flahertyet al, N Engl J Med 363:809-819, 2010). Although the significantclinical activity of BRAF selective inhibitors is a major breakthroughin the treatment of this disease, there are still hurdles to overcome tooptimize this targeted therapy approach (Flaherty and McArthur, Cancer116:4902-4913, 2010). Some patients did not respond (primaryresistance). Most patients had partial responses. Moreover, the medianduration of response is approximately only 8 months (secondaryresistance). The next urgent clinical goal is to identify rationaltherapy to obtain complete response and to improve the longevity ofinitial response to BRAF-inhibitor (BRAF-I). It is disclosed herein thatantibodies that specifically bind GRP94 have a synergistic effect withBRAF inhibitors to lower viability of tumor cells and decreasemetastasis.

Example 1 Material and Methods

Generation of BRAFinhibitor (BRAF-I) PLX4720 M21 Resistant (M21R)Melanoma Cell Line.

The cell line M21 (harboring the V600E BRAF missense mutation)(4×10⁵/well) was cultured in a 6-well plate containing RPMI 1640 mediumsupplemented with 10% FBS and 2.5 μM of PLX4720. Medium was changedevery 4 days. On the 8^(th) day the concentration of PLX4720 wasincreased to 5 μM. Cells were cultured under these conditions for 12days changing the medium every 4 days. Then the concentration of PLX4720in the medium was increased to 10 μM, and the medium has been changedevery 3 days. Cells were cultured until when resistant colonies appeared(around 3 weeks). Cells were tested for resistance to BRAF-I PLX4720 byanalyzing their growth in the presence of 10 μM of PLX4720, using theMTT assay.

Effect of BRAF-I PLX4720 on GRP94 Expression by M21 Cell Line:

M21 cells were cultured in RPMI 1640 medium supplemented with 10% FBSand BIM of PLX4720. Cells were harvested after 14 days of culture, andwashed twice with PBS-BSA. Cells (1×10⁵/sample), and were stained with 1μg of anti-GRP94 mAb W9. Human IgG (HIg) and untreated cells were usedas controls. Following an incubation of 30 min at 4° C., cells werewashed three times with PBS containing 2% of BSA (PBS-BSA). Cells werethen incubated with an appropriate dilution of PE-conjugated anti-humanIgG for 30 min at 4° C. Cells were washed and fixed with 2% PFA.Immunofluorescence was measured using a Cyan cytofluorimeter.

GRP94 Expression by M21R Melanoma Cell Line:

M21 acquired resistance to BRAF-I PLX4720 following repeated exposuresto this inhibitor. BRAF-I PLX4720 resistant melanoma cells M21R andparental melanoma cells M21 were cell surface stained with theGRP94-specific mAb W9. HIg was used as negative control. The stainingwas performed as previously described Immunofluorescence was measuredusing a Cyan cytofluorimeter.

Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined with BRAF-I:

Cells (1×10⁴ cells/ml) were seeded in quadruplicate into 96-well platesand treated with W9 mAb combined with 5 μM of BRAF-I. HIg was used as acontrol. Cells growth was analyzed at different time points (1, 2, 3, 4,and 5 days) using the MTT assay.

Migration:

BRAF-I PLX4720 resistant melanoma cells M21R (2.5×10⁴/well) were seededin a 24-transwell plate (24-well insert, pore size 8 μm; BD Biosciences)in RPMI 1640 medium containing 1% FCS with W9 antibody (Ab), HIg, BRAF-Icombined with W9 Ab, BRAF-I combined with HIg, or BRAF-I only. Cellsmigrated toward RPMI1640 medium containing 1% FCS and 10 μg/mlfibronectin. After 72 hours, migrated cells were stained with HEMA 3stain set, taken picture and counted under a Zeiss Inverted FluorescenceMicroscope (AxioVision Software). Mean of six independent high powerfield (100×) were shown as columns. The experiments were performed intriplicates.

Immunoblot:

Human melanoma cell line M21R was serum starved for 3 days then seededat the concentration of 1.0×10⁵ per well in a 6-well plate in RPMI 1640medium without serum and incubated with the GRP94 mAb, HIg, untreated,BRAF-I PLX4720 (5 μM) combined with W9 mAb, BRAF-I combined with HIg, orBRAF-I only, at 37° C. for an additional 3 days. Cells were lysed inlysis buffer (10 mM Tris-HCl [pH 8.2], 1% NP40, 1 mM EDTA, 0.1% BSA, 150mM NaCl) containing 1/50 (vol/vol) of protease inhibitor cocktail(Calbiochem). Protein concentrations in the lysates were measuredutilizing the Bradford reagent (Bio-Rad, Laboratories, Hercules,Calif.). Equal amount of proteins (60 μg per well) from the clarifiedlysates were separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride(PVDF) membrane of 0.45 μm pore size (Millipore). After blocking themembranes with 5% nonfat dry milk plus 2% BSA at room temperature for 2hours, membranes were incubated overnight at 4° C. with rabbit anti-RAS,C-RAF, phosphorylated (p)-MEK(Ser217/221), ERK, p-ERK1/2(Thr202/Tyr204), PKCα and β-actin mAb (Cell Signaling Technology), mouseanti-FAK, p-FAK (Tyr397) mAb (BD Transduction Laboratories), mouseanti-calnexin mAb TO-5. Then peroxidase-conjugated secondary antibodies(anti-mouse IgG antibody, or anti-rabbit IgG antibody) were added andincubation was continued at room temperature for an additional 45minutes. Between the incubations, membranes were washed five times, 5minutes each, with PBS (pH 7.4) containing 0.1% Tween. Then boundantibodies were detected using ECL Plus Western Blotting DetectionSystem (GE Healthcare), and bands were visualized using the FOTO/AnalystInvestigator Eclipse System (Fotodyne Incorporate). The calnexin andβ-actin were used as the protein loading controls. The densities ofresultant bands were determined with ImageJ software (NIH), normalizedto that of Calnexin and β-actin, and are shown below the respectivebands. Data are expressed as the percentage of the expression inuntreated control cells.

Statistical Analysis:

Statistical analysis was performed using the t-test. Statisticalsignificance was indicated by p<0.05.

Example 2 Effect of BRAF-I PLX4720 on the Expression of GRP94 by M21Cell Line

M21 cells were cultured in RPMI 1640 medium supplemented with 10% FBSand 1 μM of PLX4720. Cells were harvested after 14 days of culture, andwashed twice with PBS-BSA. Cells (1×10⁵/sample), and were stained with 1μg of anti-GRP94 mAb W9. Human IgG (HIg) and untreated cells were usedas controls. Following an incubation of 30 min at 4° C., cells werewashed three times with PBS containing 2% of BSA (PBS-BSA). Cells werethen incubated with an appropriate dilution of PE-conjugated anti-humanIgG for 30 min at 4° C. Cells were washed and fixed with 2% PFAImmunofluorescence was measured using a Cyan cytofluorimeter and theresults show treatment of BRAF-I sensitive melanoma cell line M21 withthe BRAF-I PLX4720 results in increased expression of GRP94. No changein human IgG binding was detected (FIG. 1). These data suggest thatBRAF-I treatment enhances the anti-tumor effect of GRP94-specific W9mAb.

Example 3 GRP94 Expression by M21R Melanoma Cell Line M21

BRAF-I PLX4720 resistant melanoma cells M21R and parental melanoma cellsM21 were cell surface stained with the GRP94-specific mAb W9. HIg wasused as negative control. The staining was performed as previouslydescribed. Immunofluorescence was measured using a CYAN™cytofluorimeter. The results show that BRAF-I resistant melanoma cellsdisplay an increased expression of GRP94 when compared to the parentalM21 cells (FIG. 2). These results suggest that BRAF-I resistant melanomacells are more susceptible to the anti-tumor effect of GRP94-specificmAb.

Example 4 Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined withBRAF-I

Cells (1×10⁴ cells/ml) were seeded in quadruplicate into 96-well platesand treated with W9 mAb combined with 5 μM of BRAF-I PLX4720. HIg wasused as a control. Cells growth was analyzed at different time points(1, 2, 3, 4, and 5 days) using the MTT assay. The results show thattreatment of BRAF-I resistant M21R cell line with GRP94-specific mAb incombination with BRAF-I results in lower viability than cells treatedwith mAb W9 alone. No changes are detectable in the viability of BRAFwild type MV3 cells treated with W9 mAb alone or combined with BRAF-I(FIG. 3). This data shows the synergic anti-proliferative effect of W9mAb and BRAF-I on BRAF-I resistant cell line.

Example 5 Anti-Cell Growth Effect of GRP94-Specific mAb W9 Combined withBRAF-I

Cells (1×10⁴ cells/ml) were seeded in quadruplicate into 96-well platesand treated with W9 mAb combined with 500 nM of BRAF-I PLX4032. HIg wasused as a control. Cells growth was analyzed at different time points(1, 3 and 5 days) using the MTT assay. The results show that treatmentof M21, M21R, Colo38 and Colo38R cell line with GRP94-specific mAb incombination with BRAF-I results in lower viability than cells treatedwith mAb W9 alone (FIG. 4). This data shows the synergicanti-proliferative effect of W9 mAb and BRAF-I on BRAF-I sensitive andresistant cell line.

Example 6 Migration

BRAF-I PLX4720 resistant melanoma cells M21R (2.5×10⁴/well) were seededin a 24-transwell plate (24-well insert, pore size 8 μm; BD Biosciences)in RPMI 1640 medium containing 1% FCS with W9 antibody (Ab), HIg, BRAF-Icombined with W9 Ab, BRAF-I combined with HIg, or BRAF-I only. Cellsmigrated toward RPMI1640 medium containing 1% FCS and 10 μg/mlfibronectin. After 72 hrs incubation, as show in FIG. 5, GRP94-specificAb W9 inhibited around 20% of the motility of M21R cells towardsfibronectin in a Boyden chamber assay. When cells incubated withGRP94-specific Ab W9 combined with BRAF-I, the inhibition of migrationwas increased to 45%. This data shows the synergic anti-migration effectof W9 mAb and BRAF-I on BRAF-I resistant cell line.

Example 7 Immunoblot

In vitro incubation with the GRP94-specific W9 mAb showed a decrease inthe level of phosphorylated (p)-MEK (Ser217/221), and p-ERK1/2(Thr202/Tyr204) and p-FAK (Tyr397), in the M21R cell line (FIG. 6). Inaddition, the total protein levels of RAS, c-RAF, and PKCα were alsodecreased. Treatment of BRAF-I resistant M21R cell line with W9 incombination with BRAF-I results in lower level of RAS, c-RAF, p-MEK(Ser217/221) and p-ERK1/2(Thr202/Tyr204) than those in cells treatedwith W9 or BRAF-I alone.

Example 8 Combination Therapy with BRAF-I PLX4720 (or PLX4320) andAnti-GRP94 Antibody is More Effective than Either Agent Alone inInducing Melanoma Cell Apoptosis

To measure cell apoptosis in vitro, cells are seeded (5.0×10⁴ cells perwell) in a 96-well plate and treated with 5 different regimens asdescribed above for 2, 6 and 24 hours. Cells are then subjected tostaining simultaneously with FITC-Annexin V (green fluorescence) (BDBiosciences) and the non-vital dye propidium iodide (red fluorescence)(BD Biosciences), which allows bivariate analysis to discriminate intactcells (FITC⁻PI⁻), early apoptotic (FITC⁺PI⁻) and late apoptotic ornecrotic cells (FITC⁺PI⁺). All experiments are performed threeindependent times.

To investigate the mechanisms of action underlying the effects on cellgrowth, migration and apoptosis, cells treated as described above arelysed for examining the changes in the level of total and activatedsignaling protein molecules, such as FAK, RAS, BRAF, ERK1/2, PI3, AKT,PKCα by Western blot. The significance of the difference in cell growth,migration, apoptosis and the level of signaling molecules are analyzedusing the Student T test.

The primary resistance of melanoma cells to BRAF-I can be reversed andthe secondary resistance of melanoma cells to BRAF-I can bedelayed/prevented by targeting multiple signaling pathways with BRAF-Iand anti-GRP94 antibody. The M233^(V600E) cell line is naturally, i.e.,primarily resistant to BRAF-I (Sondergaard et al., J Transl Med 8:39,2010). In addition to M233 cell line, in order to obtain at least onemore BRAF-I primary resistant cell line, additional V600E mutated humanmelanoma cell lines are screened using a cell growth MTT assay. It isdetermined that the combination therapy can reverse their resistance toBRAF-I in these two primary resistant cell lines in cell growth assays.Furthermore, the cell lines M21^(V600E) and SK-MEL-5^(V600E) are used,both of which are BRAF-I sensitive to confirm that secondary resistancecan be delayed or prevented in the presence of anti-GPR94 in cell growthassays.

Cells were starved for 12 hours and seeded at a density of 2×10⁵/ml in a6-well plate and treated for 6 hours with BRAF-I PLX4032 (500 nM) andGrp94-specific mAb W9 (20 μg/ml) in RPMI 1640 medium plus 1.5% FCS.Cells were stained with Annexin V-FITC and PI, and evaluated forapoptosis by flow cytometry according to the manufacturer's protocol (BDPharMingen, San Diego, Calif., USA). The early apoptotic cells (annexinV-positive, PI-negative) were determined using a flow cytometer. Theresults show that treatment of M21 and M21R cell lines withGRP94-specific mAb in combination with BRAF-I PLX4032 induced more cellapoptosis than cells treated with mAb W9 and BRAF-I PLX4032 alone (FIG.7). This data shows the synergic pro-apoptpsis effect of W9 mAb andBRAF-I on BRAF-I sensitive and resistant cell line.

Example 9 Combination Therapy with BRAF-I PLX4032 and Anti-GRP94Antibody is More Effective than Either Agent Alone in Inhibition ofCancer Stem Cell Proliferation in Vitro

Cells growing in the exponential phase were seeded at a density of2×10⁵/ml. The cells were treated for 3 days with BRAF-I PLX4032 (500 nM)and Grp94-specific mAb W9 (20 μg/ml) in RPMI 1640 medium plus with 1.5%FCS. Cells were stained with ALDEFLUOR® according to the manufacturer'sprotocol (Stem Cell Technologies). Incubation of cells with ALDEFLUOR®in the presence the ALDH1-specific inhibitor diethylaminobenzaldehyde(DEAB) was used as a negative staining control for the assay. Then cellswere stained with ABCB5-specific mAb RK1(1 μg/ml) for 30 min at 4° C.,and incubated with APC-conjugated secondary mAb (1:200) (JacksonImmunoresearch). The results show that treatment of M21 and M21R celllines with GRP94-specific mAb in combination with BRAF-I PLX 4032results in lower viability than cells treated with mAb W9 and PLX4032alone (FIG. 8). This data shows the synergic inhibition of growth ofcancer stem cell effect of W9 mAb and BRAF-I on BRAF-I sensitive andresistant cell line.

Example 10 Inhibition by Grp94-Specific mAb W9 and BRAF-I PLX 4032 ofSignaling Pathways in BRAFV600E Mutant and BRAF-I Resistant MelanomaCells

Cells growing in the exponential phase were seeded at a density of2×10⁵/ml. The cells were treated for 3 days with BRAF-I PLX4032 (500 nM)and Grp94-specific mAb W9 (5 μg/ml) in RPMI 1640 medium plus with 2%FCS. Then the cells were collected and lysed in lysis buffer. Theexpression and activation of multiple signaling molecules were analyzedby immunoblot. The results show that the combination of GRP94-specificmAb W9 and BRAF-I PLX4032 inhibited the expression and activation ofsignaling molecules important for cell proliferation (RAS, MER, ERK1/2),for hedgehog signaling pathway (GLI1 and SHh) as compared to the proteinlevel in cells treated by GRP94-specific mAb W9 and BRAF-I PLX4032 alone(FIG. 9).

Example 11 V600E Mutated Human Melanoma Cell Lines and Resistance toPLX4720 and/or PLX4320

Several V600E mutated human melanoma cell lines are tested to identifythe cell lines which maintain the same growth rate at days 1, 3 and 7 inthe presence and absence of BRAF-I PLX4720 (or PLX4320) at 5 μM in MTTassays. The identified cell lines, which are primary resistant to BRAF-Iare confirmed with a higher dose (10 μM) of PLX4720 (or PLX4320) in cellgrowth assays.

The cells are treated with treatment with BRAF-I PLX4720 and an antibodythat specifically binds GRP94 (anti-GRP94) to determine that primaryresistance of human melanoma cell lines to BRAF-I PLX4720 (or PLX4320)is affected by the combination therapy. In addition to cell lineM233^(V600E), the additional primary resistant cell lines are treated inseveral different regimens and then cell growth, migration and apoptosisis measured. Combination therapy affects the growth, migration andapoptosis of the cell lines.

It is also determined that combination treatment with BRAF-I PLX4720 (orPLX4320) and anti-GRP94 delays and/or prevents secondary resistance ofhuman melanoma cell lines to BRAF-I PLX4720 (or PLX4320). It has beendetermined that V600E mutated melanoma cell lines, which are sensitiveto BRAF-I in vitro treatment, became resistant, i.e., secondaryresistant, to PLX4720 (or PLX4320) after 3-4 week of in vitro treatmentwith increasing doses (2.5-10 μM) of PLX4720 (or PLX4320).

The cells (4×10⁵/well) are cultured in a 6-well plate containing 2 ml ofRPMI 1640 medium supplemented with 10% FBS and 2.5 μM of PLX-4720 (orPLX4320). Medium is changed every 4 days. On day 8, the dose of PLX4720(or PLX4320) is increased to 5 μM. Cells are cultured under theseconditions for 12 days changing the medium every 4 days. Then the doseof PLX4720 (or PLX4320) is increased to 10 μM, changing the medium every3 days. Cells are cultured until resistant colonies appear (around 3-4weeks). Cells are tested for resistance to BRAF-I by testing theirgrowth in the presence of 10 μM of PLX4720 (or PLX4320), using the MTTassay. Simultaneously, cells in one well will be set up in the exactsame way except adding anti-GRP94 every 3-4 days at its optimal doseidentified as described above. In the control well, everything is notchanged but only anti-GRP94 is replaced with the isotype control mAb.The cells, in the presence of both PLX4720 (or PLX4320) and anti-GRP94,are kept in culture as long as needed for monitoring whether and/or whensecondary resistance occurs.

To analyze the mechanisms of action of combination treatment in primaryand secondary resistances, cells are lysed for examining the changes inthe level of total and activated signaling protein molecules by westernblot as above. The significance of the difference in cell growth and thelevel of signaling molecules is analyzed using the Student T test.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A method of treating a subject diagnosed with a tumor thatexpresses a BRAF mutation, comprising administering to the subject (1) atherapeutically effective amount of an monoclonal antibody or antigenbinding fragment thereof that specifically binds glucose regulatedprotein (GRP) 94; and (2) a therapeutically effective amount of a BRAFinhibitor, thereby treating the tumor in the subject.
 2. The method ofclaim 1, wherein the tumor is a breast cancer, prostate cancer, ovariancancer, colon cancer, stomach cancer, pancreatic cancer, glioma,chordoma, chondrosarcoma, thyroid cancer, colon cancer, glioma, renalcancer, lung cancer, bladder cancer, non-Hodgkin's lymphoma, or asquamous cell carcinoma, wherein cells of the tumor express GRP94. 3.The method of claim 2, wherein the squamous cell carcinoma is head andneck carcinoma, lung carcinoma, prostate carcinoma, esophagus carcinoma,vagina carcinoma or cervix carcinoma.
 4. The method of claim 1, whereinthe subject has primary or secondary resistance to the BRAF inhibitor.5. The method of claim 1, wherein the BRAF inhibitor is PLX4032 orPLX4720.
 6. The method of claim 1, comprising administering to thesubject a therapeutically effective amount of an antigen bindingfragment of a monoclonal antibody that specifically binds GRP94.
 7. Themethod of claim 1, wherein the monoclonal antibody or antigen bindingfragment comprises a heavy chain variable domain, and wherein the heavychain variable domain of the monoclonal antibody comprises the aminoacid sequence set forth as amino acids 26-33 of SEQ ID NO: 3, aminoacids 51-58 of SEQ ID NO: 3, and amino acids 97-103 of SEQ ID NO:
 3. 8.The method of claim 5, wherein the monoclonal antibody or antigenbinding fragment comprises a light chain variable domain, wherein thelight chain variable domain of the antibody comprises the amino acidsequence set forth as amino acids 27-32 of SEQ ID NO: 4, amino acids50-52 of SEQ ID NO: 4, and amino acids 89-97 of SEQ ID NO:
 4. 9. Themethod of claim 1, wherein treating the tumor comprises decreasing themetastasis of the tumor.
 10. The method of claim 1, wherein the subjectis human.
 11. The method of claim 1, further comprising administeringone or more additional chemotherapeutic agents.
 12. The method of claim11, wherein the one or more additional chemotherapeutic agents comprisesan alkylating agent, a topoisomerase inhibitor, a platinum anti-cancerdrug, or a combination thereof.
 13. The method of claim 12, wherein thealkylating agent comprises a nitrosourea or a taxane.
 14. The method ofclaim 1, wherein the therapeutically effective amount of the BRAFinhibitor and the therapeutically effective amount of the antibody orantigen binding fragment thereof that specifically binds glucoseregulated protein (GRP) 94 are administered simultaneously.
 15. Themethod of claim 1, wherein cells in the tumor comprise a BRAF mutation.16. The method of claim 15, wherein the BRAF mutation is a V600Emutation.
 17. The method of claim 15, wherein the BRAF mutation is aR462I, I463S, G464E, G464V, G466A, G466E, G466V, G469A, G469E, N581S,E585K, D594V, F595L, G596R, L597V, T599I, V600D, V600E, V600K, V600R,K601E or A728V mutation.
 18. The method of claim 15, further comprisingdetecting a V600E BRAF mutation in a sample from the subject, whereinthe sample comprises cells from the tumor.
 19. The method of claim 1,wherein: the BRAF inhibitor is PLX4032 or PLX4720; and the monoclonalantibody, or antigen binding fragment thereof comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain of the monoclonal antibody comprises the aminoacid sequence set forth as amino acids 26-33 of SEQ ID NO: 3, aminoacids 51-58 of SEQ ID NO: 3, and amino acids 97-103 of SEQ ID NO: 3 andthe light chain variable domain of the antibody comprises the amino acidsequence set forth as amino acids 27-32 of SEQ ID NO: 4, amino acids50-52 of SEQ ID NO: 4, and amino acids 89-97 of SEQ ID NO: 4.